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Am J Physiol Endocrinol Metab 293: E1012-E1020, 2007. First published July 24, 2007; doi:10.1152/ajpendo.00649.2006
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The effect of fat removal on glucose tolerance is depot specific in male and female mice

Haifei Shi,1 April D. Strader,2 Stephen C. Woods,1 and Randy J. Seeley1

1University of Cincinnati, Cincinnati, Ohio; and 2Southern Illinois University School of Medicine, Carbondale, Illinois

Submitted 28 November 2006 ; accepted in final form 19 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Energy is stored predominately as lipid in white adipose tissue (WAT) in distinct anatomical locations, with each site exerting different effects on key biological processes, including glucose homeostasis. To determine the relative contributions of subcutaneous and visceral WAT on glucose homeostasis, comparable amounts of adipose tissue from abdominal subcutaneous inguinal WAT (IWAT), intra-abdominal retroperitoneal WAT (RWAT), male gonadal epididymal WAT (EWAT), or female gonadal parametrial WAT (PWAT) were removed. Gonadal fat removal in both male and female chow-fed lean mice resulted in lowered glucose levels across glucose tolerance tests. Female lean C57BL/6J mice as well as male and female lean FVBN mice significantly improved glucose tolerance, indicated by decreased areas under glucose clearance curves. For the C57BL/6J mice maintained on a high-fat butter-based diet, glucose homeostasis was improved only in female mice with PWAT removal. Removal of IWAT or RWAT did not affect glucose tolerance in either dietary condition. We conclude that WAT contribution to glucose homeostasis is depot specific, with male gonadal EWAT contributing to glucose homeostasis in the lean state, whereas female gonadal PWAT contributes to glucose homeostasis in both lean and obese mice. These data illustrate both critical differences among various WAT depots and how they influence glucose homeostasis and highlight important differences between males and females in glucose regulation.

intra-abdominal fat; subcutaneous fat; adipokine; portal drainage

ENERGY IS PREDOMINATELY STORED AS LIPID in white adipose tissue (WAT), and WAT from different anatomical locations has been implicated in the regulation of a wide array of metabolic parameters. Abdominal obesity with excessive subcutaneous abdominal and intra-abdominal WAT, for example, is associated with increased insulin resistance. One hypothesis is that intra-abdominal WAT has a disproportionately adverse effect on glucose homeostasis (7, 10, 27, 42, 44), and this concept is supported by several human studies (12, 15, 37) but challenged in others (1, 2, 18, 30) that suggest that subcutaneous abdominal WAT is more closely related with insulin resistance.

The surgical removal of WAT, i.e., by liposuction or lipectomy, has been used to study the relationship between abdominal obesity and insulin resistance in humans and rodents. Liposuction of obese women that causes a sizable reduction of subcutaneous abdominal fat results in no improvement in insulin sensitivity, plasma glucose, or insulin levels (25). Improvement of insulin resistance after removal of certain WAT depots in rat obese models provides direct evidence for the causal role of these fat pads in causing insulin resistance. For example, simultaneous removal of both gonadal epididymal WAT (EWAT) and intra-abdominal perinephric WAT in aging obese male rats leads to marked reductions of insulin resistance (5, 16). Men and women distribute body fat differently, with females carrying relatively more subcutaneous fat and males having a greater percentage of visceral fat (22, 26, 44), and men have a greater risk for incurring obesity-related metabolic disorders. This makes it imperative to understand how specific fat depots might contribute to glucose homeostasis and other metabolic parameters and whether males and females differ in fundamental ways.

The goal of the current study was to identify the contribution of specific individual WAT depots on glucose homeostasis in adult male and female lean and moderately obese mice fed a high-fat butter-based diet (HFD). We assessed glucose tolerance before and after removing a comparable amount of abdominal subcutaneous inguinal WAT (IWAT), intra-abdominal retroperitoneal WAT (RWAT), male gonadal EWAT, or female gonadal parametrial WAT (PWAT). Because adipose tissues at different locations are quite different in size, we removed equivalent amounts of adipose tissue within each sex and diet cohort as opposed to whole fat pads; thus the effects and contribution from individual adipose tissue locations could be directly compared. RWATs are smaller in females than males, whereas total body fat levels are comparable between males and females. Thus, a smaller percentage of total fat from females than from males was removed. As a consequence, all direct comparisons are made within each sex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Cohorts of 12-wk-old male and female FVBN mice and 12- and 8-wk-old male and female C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Both strains are commonly used in obesity and diabetes studies. All mice were individually housed in microisolator cages in pathogen-free rooms with temperature and humidity controlled and a 12:12-h light-dark cycle with lights on at 0500 and off at 1700. The 12-wk-old FVBN and C57BL/6J mice had ad libitum access to regular pelleted rodent chow (rodent diet 8604: 3.4 kcal/g, 12% fat; Harlan Teklad), and the 8-wk-old C57BL/6J mice had ad libitum access to a HFD (4.54 kcal/g, 40% fat; Research Diets, New Brunswick, NJ) for 5 wk prior to the experimental procedures. We included HFD-fed C57BL/6J but not FVBN mice in the current study because C57BL/6J mice are particularly susceptible to diet-induced obesity, whereas FVBN mice are not. All chow-fed mice received an intraperitoneal glucose tolerance test (IPGTT) and an intraperitoneal insulin tolerance test (IPITT) 1 and 2 wk before and 2 and 3 wk after either sham or lipectomy surgeries. HFD-fed mice received an IPGTT 1 wk before and 2 wk after surgeries. The time points of postsurgical 2–3 wk were chosen because the animals had recovered from the surgery and resumed normal behavior, and body fat had not compensatorily increased, at that time. All animal procedures were approved by the University of Cincinnati Institutional Animal Care and Use Committee.

Determination of cyclicity. Vaginal smears were performed immediately after IPGTT and IPITT in intact chow-fed female C57BL/6J mice and were stained with a DipQuick staining kit (Jorgensen Laboratories, Loveland, CO) for the determination of the phase of the estrous cycle based on the pattern of cell types of smear samples (6).

Surgical procedures. Animals were anesthetized by an intraperitoneal injection of avertin (200 mg/kg). The hair was removed from the incision area, and the area was then sterilized with a 70% ethanol- and betadine-soaked gauze. For IWAT lipectomy (IWATx) surgeries, short (~1.0 cm) skin incisions were made bilaterally, beginning at the lateral side of the proximal end of the hindlimb and continuing rostrally and ventrally so that the abdominal subcutaneous IWAT was exposed bilaterally. For RWAT lipectomy (RWATx), skin and muscle incisions were made bilaterally on the dorsal side of the animal and parallel to the spinal column so that intra-abdominal RWAT was exposed. For male EWAT or female PWAT lipectomy surgeries (EWATx or PWATx, respectively), a single abdominal midline incision was made through which bilateral EWAT or PWAT pads could be accessed. Care was taken with the depth of all incisions to avoid disturbing the underlying blood vessels, musculature, and organs. Approximately equivalent amounts of adipose tissue were carefully dissected (Table 1) without adjacent muscle tissue or organs being damaged. Specific care was taken to avoid damage to the testicular blood supply for EWATx surgeries, and the most rostral portion of EWAT relative to the testes was taken. PWAT at the level of the oviduct, distal to the ovaries, and adjacent to the uterus was removed in PWATx surgeries. PWAT was lifted before dissection, and no deep incision was involved; thus the PWATx procedure caused little or no bleeding. Normal estrous cycles were maintained in all females before and after the surgeries in the current study, indicating normal female gonadal function following surgeries. As shown in Table 1, smaller percentages of adipose tissue were removed in the female mice than the males within the same cohort. This is due to the fact that the RWAT is smaller in females, whereas total body fat levels were less different but comparable between males and females. RWAT is considerably smaller than IWAT and gonadal adipose tissues. Therefore, as much RWAT as possible was removed without surrounding organs, muscles, or vessels being damaged, and part of IWAT or gonadal WAT was removed to match the amount of fat removed from each group.


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  		Table 1. Amount of adipose tissue removed from chow- or HFD-fed male or female mice

 
One-third of the sham-operated animals received a similar procedure as IWATx, RWATx, or E/PWATx, respectively, in that the relevant WAT depot was visualized but not removed (sham). Specifically, for IWAT sham surgeries, the incision was made and the inguinal fat pad localized, but there was no further dissection or cutting. For RWAT sham surgeries, the incision was made and the muscle incised so that RWAT could be visualized, but the fat was not manipulated. For E/PWAT sham surgeries, a pair of tissue forceps was used to raise the tip of the E/PWAT pad, and then the pad was returned to its original location. Because there were no differences among these sham control groups, their data were combined for the analyses. After all surgeries, the abdominal peritoneum was sutured with sterile absorbable vicryl sutures, and the skin was closed with sterile wound clips.

IPGTT and IPITT. IPGTT and IPITT were performed during the light phase according to previously established procedures (40). Mice were fasted overnight for 16 h, and all blood samples were obtained from the tip of the tail vein of freely moving mice. After a baseline blood sample was taken (0 min), 1.5 g/kg of 20% D-glucose (Phoenix Pharmaceutical, St. Joseph, MO) or 1 U/kg body wt of insulin (Novolin; Novo Nordisk) was injected. Subsequent blood samples were taken at 15, 30, 45, and 60 min after glucose or insulin administration, and glucose was measured on duplicate samples using FreeStyle glucometers and test strips (FreeStyle, Alameda, CA). An additional blood sample was taken from the tail vein 13–15 min after the glucose administration for measurement of plasma insulin using the rat insulin enzyme-linked immunosorbent assay kits (Crystal Chem, Downers Grove, IL). The coefficients of variation of intra-assay and interassay are 5.5 and 6.1%, respectively. To assess glucose tolerance, calculations of the area under glucose curves (AUC) were made on the basis of the glucose baseline levels at 0 min. To assess insulin sensitivity, slope of change of glucose levels representing the rate of disappearance of glucose following insulin administration between baseline 0 and 45 min was calculated.

Statistical analyses. Data are expressed as means ± SE. Comparisons among multiple groups were made using one-way analysis of variance. Post hoc tests of individual groups were made using Tukey's tests (SigmaStat 3.1, San Rafael, CA). Significance was set at P < 0.05. Exact probabilities and test values were omitted for simplicity and clarity of the presentation of the results.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IPGTT and IPITT of intact female mice. We performed IPGTT and IPITT on 40 intact female C57BL/6J mice and examined their phases of the estrus cycle immediately after the tests. Glucose levels at all time points of the IPGTTs and the AUCs were comparable among all phases (proestrus: n = 5, estrus: n = 5, metestrus: n = 18, and diestrus: n = 12; Fig. 1A). Specifically, AUCs were 8,690.25 ± 1,507.61 for proestrus, 7,947.75 ± 797.12 for estrus, 9,036.04 ± 945.14 for metestrus, and 8,587.19 ± 1,149.82 for diestrus. In addition, glucose levels at all time points of the IPITT and slopes of the change of glucose levels indicated by the glucose disappearance rates between baseline 0 and 45 min after insulin injections were also comparable among all phases (proestrus: n = 3, estrus: n = 19, metestrus: n = 13, and diestrus: n = 5; Fig. 1B). Specifically, glucose disappearance rates were 2.07 ± 0.53 for proestrus, 1.88 ± 0.17 for estrus, 1.85 ± 0.27 for metestrus, and 1.89 ± 0.37 for diestrus. Therefore, although estrogen regulates energy balance, estrogen within the physiological range experienced during different stages of the estrus cycle did not affect glucose tolerance or insulin sensitivity measured by IPGTT or IPITT in mice in this paradigm. Because glucose tolerance or insulin sensitivity was not affected by different phases of the estrus cycle, the postsurgical IPGTT and IPITT were performed exactly 2 and 3 wk, respectively, following surgery regardless of the phases of the estrus cycle.


Figure 1
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  		Fig. 1. Intraperitoneal glucose tolerance tests and intraperitoneal insulin tolerance tests of chow-fed intact female C57BL/6J mice. A: glucose tolerance was not different across estrous cycle; proestrus ( n = 5), estrus ( n = 5), metestrus (n = 18), and diestrus (n = 12). B: insulin sensitivity was not different across estrous cycle; proestrus (n = 3), estrus (n = 19), metestrus (n = 13), and diestrus (n = 5).

 
Statistical power analysis. A power analysis was conducted by using an effect size of 35% for the AUC and the standard deviation (SD) from our sham group, a group size of 9, and {alpha} as 0.05. Specifically, for male C57BL/6J chow-fed cohort with difference in means as 2,500 and SD as 1,500, the power is 0.812; for female C57BL/6J chow-fed cohort with difference in means as 3,000 and SD as 1,500, the power is 0.941. For male FVBN chow-fed cohort with difference in means as 1,800 and SD as 1,200, the power is 0.710; for female FVBN chow-fed cohort with difference in means as 2,000 and SD as 1,200, the power is 0.812. For male C57BL/6J HFD-fed cohort with difference in means as 2,000 and SD as 800, the power is 0.996; for female C57BL/6J HFD-fed cohort with difference in means as 1,500 and SD as 800, the power is 0.904. Thus, the design of current study was well powered to find reasonable effect sizes. Although the possibility for missing some of effects cannot be eliminated, those effects are relatively small.

Chow-fed mice. Sham male C57BL/6J and FVBN mice had similar pre- and postsurgical glucose clearance curves, with comparable glucose levels at all time points during the IPGTT (Figs. 2A and 3A). The mean baseline glucose level of the chow-fed male C57BL/6J mice was lower after sham surgery (Table 2). Chow-fed female C57BL/6J sham mice likewise had similar glucose parameters at all time points before and after glucose injection between the two tests (Fig. 2B). Female FVBN sham mice had elevated glucose before and 15 min after glucose injection post- relative to presurgery, and the levels during the two tests were comparable after that (Fig. 3B). The AUCs and the 13- to 15-min insulin levels after glucose injection were similar in pre- and postsurgical sham males and females (Tables 2 and 3). Thus, IPGTTs conducted 3 wk apart did not differ in C57BL/6J or FVBN sham-operated lean mice. Neither sham surgical procedure nor 3-wk chow-fed period affected glucose tolerance significantly. As such, the pre- and postsurgical IPGTTs were compared within groups for the lean mice.


Figure 2
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  		Fig. 2. Intraperitoneal glucose tolerance tests of chow-fed sham or epididymal/parametrial white adipose tissue lipectomy (E/PWATx) male or female C57BL/6J mice. Neither chow-fed C57BL/6J male (A; n = 8) nor female (B; n = 9) mice changed glucose tolerance after sham surgery. Both chow-fed C57BL/6J male (C; n = 9) and female (D; n = 10) mice had lower glucose levels after having gonadal WAT removed (EWATx in males or PWATx in females). *P < 0.05.

 

Figure 3
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  		Fig. 3. Intraperitoneal glucose tolerance tests of chow-fed sham or E/PWATx male or female FVBN mice. Neither chow-fed FVBN male (A; n = 12) nor female (B; n = 8) mice changed glucose tolerance after sham surgery. Both chow-fed FVBN male (C; n = 9) and female (D; n = 10) mice had lower glucose levels after having gonadal WAT removed (EWATx in males or PWATx in females). *P < 0.05.

 

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  		Table 2. Pre- and postsurgical IPGTT of chow-fed male or female C57BL/6J mice

 

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  		Table 3. Pre- and postsurgical IPGTT of chow-fed male or female FVBN mice

 
Following EWATx, male C57BL/6J mice had significantly decreased glucose both at baseline and at 15, 30, and 45 min after exogenous glucose administration (Fig. 2C). The AUC, calculated on the basis of baseline glucose levels, although decreased after surgery, was not significantly different between pre- and post-EWATx C57BL/6J mice (Table 2). Thus, the glucose curves from IPGTTs of pre- and post-EWATx C57BL/6J mice differed, but AUC did not change correspondingly due to the significant decrease of baseline fasting glucose following EWATx. The 13- to 15-min plasma insulin level of post-EWATx males was similar as that of pre-EWATx group, but it was significantly lower than that of the postsham mice (Table 2).

Male FVBN EWATx mice had significantly decreased glucose levels at 15 and 30 min after exogenous glucose administration as well as a lower AUC (Fig. 3C and Table 3), suggesting improved glucose tolerance after EWATx in chow-fed FVBN males. The 13- to 15-min plasma insulin level was significantly higher following EWATx than before (Table 3). Thus, male lean FVBN mice had improved glucose tolerance after EWATx that was associated with increased insulin.

Female C57BL/6J PWATx mice had comparable pre- and postsurgical baseline glucose levels, with decreased glucose levels at all time points following exogenous glucose (Fig. 2D) and a significantly lower AUC after glucose (Table 2), but it was not accompanied by an altered insulin level (Table 2), suggesting increased insulin sensitivity compared with the presurgical condition. Female FVBN PWATx mice also had decreased glucose levels (at 45 min) and a lower AUC after glucose (Fig. 3D), but it was not accompanied by an altered insulin level (Table 3), suggesting increased insulin sensitivity compared with the presurgical females.

Removal of a similar amount of intra-abdominal RWAT or abdominal subcutaneous IWAT did not change glucose levels after glucose injections, AUCs, or 13- to 15-min insulin levels during IPGTT in either sex (Tables 2 and 3), although male C57BL/6J RWATx and IWATx mice and female C57BL/6J IWATx mice had reduced fasting glucose levels after surgery (Table 2). It is noteworthy that all postsurgical male C57BL/6J mice had decreased baseline fasting glucose levels compared with their male presurgical counterparts, whereas only female C57BL/6J post-IWATx group significantly decreased baseline glucose levels.

No difference in the IPITT among any different lipectomy groups and their sham surgical controls was found in the current study. Specifically, all groups had comparable glucose disappearance rates indicated by the slopes of the change of glucose level following insulin injection before and after surgeries (Table 4). In addition, the rates of disappearance of glucose following insulin administration were comparable among treatment and sham groups (Table 4).


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  		Table 4. Pre- and postsurgical IPITT of chow-fed male or female C57BL/6J or FVBN mice

 
HFD-fed mice. Mice were maintained on the HFD throughout the study. Both male and female mice fed the HFD had a significantly increased glucose AUC following sham surgeries (Table 5), suggesting that increased time spent consuming the HFD worsened glucose tolerance, as reported by others (9, 17, 43). In addition, female mice had higher glucose levels at 30, 45, and 60 min during IPGTT after sham surgery (Fig. 4B). Because the cumulative worsening of glucose tolerance would mask any improvement of glucose tolerance caused by fat removal, we compared postsurgical glucose tolerance between sham and surgical groups, carefully matching mice in the various groups with their presurgical AUC and fasting glucose (Table 5).


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  		Table 5. Pre- and postsurgical IPGTT of HFD-fed male or female C57BL/6J mice

 

Figure 4
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  		Fig. 4. Intraperitoneal glucose tolerance tests of high-fat butter-based diet (HFD)-fed sham or E/PWATx male or female C57BL/6J mice. Both HFD-fed C57BL/6J male (A; n = 9) and female (B; n = 10) mice developed glucose intolerance after sham surgeries. C: male mice had similar glucose tolerance after EWATx surgery (n = 10) than sham males (n = 9). D: female mice had better glucose tolerance after PWATx surgery (n = 9) than sham females (n = 10). *P < 0.05.

 
Male EWATx mice had similar glucose clearance curves (Fig. 4C), AUC, and 13- to 15-min insulin levels as male sham mice (Table 5). In contrast, female PWATx mice had lower glucose levels (Fig. 4D) and AUC than their sham controls (Table 5) but similar insulin levels (Table 5), suggesting improved glucose tolerance due to increased insulin sensitivity. Consistent with the lean subjects, obese male and female RWATx or IWATx mice had similar IPGTT parameters as sham mice (Table 5).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There is a well-documented association between the accumulation of abdominal fat and insulin resistance. Human studies attempting to identify which of the various abdominal depots is most closely related to insulin resistance have led to different conclusions. Some studies (7, 10) found that increased intra-abdominal adiposity is a risk factor for insulin resistance. Others (1, 2, 18, 30) reported that subcutaneous truncal fat plays a major role in obesity-related insulin resistance, with only a weak correlation between insulin resistance and intraperitoneal and retroperitoneal fat levels. An important goal of the current studies was to identify a possible causal role for specific fat pads by selective surgical manipulation and subsequent assessment of glucose tolerance.

We examined the relative contribution of several WAT depots to glucose homeostasis by surgically removing equivalent amounts of adipose tissue from subcutaneous IWAT, intra-abdominal RWAT, gonadal EWAT, or PWAT. We found that glucose tolerance was improved following partial gonadal fat removal in both male and female mice fed a low-fat chow diet. In contrast, EWATx did not improve glucose tolerance in mice fed a HFD. Female mice with PWATx remained glucose tolerant after prolonged exposure to a HFD. The removal of comparable amounts of IWAT or RWAT did not affect glucose tolerance in either dietary condition. PWAT has rarely been studied, perhaps because few studies explicitly include females. The present data therefore make a strong case for PWAT as a major influence on glucose homeostasis.

No difference in the IPITT among any different lipectomy groups and their sham surgical controls was found in the current study. Although these data may suggest no change of insulin sensitivity following fat removal, negative IPITT data have a number of limitations in their interpretation. In particular, IPITT is a relatively insensitive measure of insulin sensitivity. Failure to find differences does not mean that differences in insulin sensitivity might not contribute to the differences observed in glucose tolerance. In addition, the peak insulin level of chow-fed post-EWATx C57BL/6J males was significantly lower than that of the postsham mice within the same cohort (Table 2). Relatively low insulin level from IPGTTs of post-EWATx C57BL/6J mice may suggest improved insulin sensitivity or decreased pancreatic insulin secretion following EWATx. The peak insulin level of male FVBN EWATx mice, however, increased following surgery (P = 0.05; Table 3), suggesting increased insulin secretion. One of definitive ways to determine whether insulin sensitivity is altered would be to conduct a hyperinsulinemic-euglycemic clamp experiment. Such clamp experiments are quite challenging in mice and would require significant effort given the number of different groups required for these experiments. Although studying insulin action following glucose challenge was beyond the scope of the current study, future work using such technique is necessary to determine the components of glucose tolerance that are differentially sensitive to the effects of the removal of different fat depots.

The weights of the RWAT are smaller in females, whereas total body fat levels are comparable between males and females. As a consequence, smaller percentages of adipose tissue were removed in the female mice than the males in the current study. Although direct comparisons within but not between each sex were made, it is noteworthy that female mice with smaller percentages of adipose tissue removed than males had more remarkable improvement of glucose tolerance after gonadal fat removal, suggesting that more impressive improvement would occur if a similar percentage of adipose tissue was removed in females.

Visceral fat is also implicated in metabolic control. The removal of a combination of EWAT and perinephric WAT was previously reported to cause metabolic improvement in aging or diabetic rats (5, 16), and partial removal of gonadal WAT in the current study significantly improved glucose tolerance. No combination of two different fat pads was removed in the current study since the effect of individual fat removal was the primary goal. The amount of fat removed from previous reports was considerably larger than in the current study because we were seeking to remove comparable amounts of fat from each group within each cohort. Significant improvements were nonetheless observed, although the consequence was not as large as previous reports in which the entire fat pad was removed.

It was previously reported that removal of subcutaneous fat does not improve insulin action or sensitivity in humans (25) or rats (16). Our data using mice corroborate these findings. One report (45) indicated that lipectomy of subcutaneous fat in conjunction with maintenance on a HFD caused hypertriglyceridemia, hyperinsulinemia, and increased fat content in liver in female Syrian hamsters. In that study, glucose tolerance was examined 11 wk after lipectomy, when total body fat had recovered. Those hamsters had elevated intra-abdominal fat, serum triglycerides, and liver fat content (45). Because we explicitly did not want to wait until all compensation for the lost fat had occurred, we examined glucose tolerance 2 wk after surgery to avoid effects from compensatory growth of adipose tissue in other anatomic sites. As expected, glucose levels, areas under curves, and peak insulin levels were not significantly changed following partial subcutaneous inguinal fat removal in the current study.

Two hypotheses exist regarding the mechanism for differential contribution of distinct WAT depots to glucose homeostasis: the portal drainage hypothesis and the differential adipokine secretion hypothesis. Björntorp (8) argued that the determining factor is based on venous drainage. Visceral WAT, including omental and mesenteric WAT, is drained by the portal circulation. Metabolic products of visceral adipose tissue, including nonesterified free fatty acids and glycerol, reach the liver directly and thus could exert disproportionately greater effects on hepatic metabolism and hepatic insulin resistance (8). The portal drainage hypothesis is also consistent with the failure of human (subcutaneous) liposuction (11, 25) and IWATx in the current mouse study to improve glucose tolerance under either chow or the HFD.

None of the WAT depots that were manipulated in the current study is considered portal visceral WAT. Venous drainage of IWAT, RWAT, and E/PWAT are all via the inferior vena cava. This is a possible explanation for the lack of effect on glucose tolerance after fat removal in HFD-fed male mice. However, our observation that PWATx improves glucose tolerance in female mice suggests that, in addition to a possible role that hepatic portal drainage might have in mediating the effects of fat on glucose homeostasis, at least one nonportal adipose depot is also important.

Different WAT depots contain adipocytes with distinct intrinsic characteristics, including receptors, adipokines, and transcription factors (4). The specific cocktail of adipokines released (including leptin, adiponectin, resistin, and many others) can modulate glucose homeostasis via actions on the brain, liver, muscle, or other tissues. Therefore, it is feasible that the removal of certain WAT adipocytes could potentially alter the circulating amounts or relative rations of these factors, some of which have deleterious effects on glucose homeostasis. Glucose tolerance was improved following removal of a small amount of gonadal adipose tissue that did not impact total adiposity. It is possible that levels of adipokines such as adiponectin or metabolites such as nonesterified fatty acids could be changed. Future study focuses on investigating changes in glucose homeostasis- and adipose tissue metabolism-related endocrine and paracrine factors would contribute to determine the mechanism for this phenomenon.

Adipokine gene expression varies as a function of WAT depot and sex. Many adipokines are highly expressed in gonadal WAT in rodents. For example, the level of leptin mRNA is higher in gonadal and perirenal WAT than in omental and subcutaneous WAT in mice (41), whereas in rats leptin mRNA expression is higher in gonadal WAT than in subcutaneous and perirenal WAT (21). In contrast to the leptin expression in rodents, leptin mRNA is more highly expressed in subcutaneous IWAT compared with visceral WAT in humans (31, 32), and plasma leptin levels are higher in women than in men with the same body mass index (35). Other adipokines, like adiponectin and resistin, are relatively overexpressed in perinephric and EWAT compared with IWAT in male rats (16). In addition, resistin mRNA levels are the highest in female gonadal fat (19, 39). Because increased resistin is implicated in insulin resistance (39), depot-specific reductions in resistin secretion could contribute to the improvement in glucose tolerance following gonadal fat removal, including female PWATx in the present study.

Adipocytes from different WAT depots have been reported to have dissimilar glycolytic and lipolytic activities. If all small and large adipoctyes are considered, RWAT adipocytes are metabolically similar to those of EWAT, and both have markedly greater insulin-stimulated glucose metabolism than the adipocytes of subcutaneous WAT (14). Another study (34) reported that insulin-stimulated glucose metabolism per cell is higher in large EWAT or RWAT adipocytes from older rats compared with smaller ones from younger rats. In summary, adipocytes of EWAT have greater lipolytic activity than those of RWAT. In addition, adipocytes of both EWAT and RWAT are more metabolically active than those of subcutaneous IWAT, as measured by in vivo microdialysis technique in rats (13, 36). The improvement of glucose tolerance following removal of EWAT in lean mice, but not RWAT or IWAT, could be due to the greater glycolytic and lipolytic activity of EWAT adipocytes from the current study.

Gonadal fat depot removal procedures (i.e., EWATx or PWATx) were carefully performed to avoid bleeding. Nevertheless, our previous EWATx study reported that the weights of testis from EWATx hamsters decreased significantly compared with sham-operated males, although circulating testosterone level was not affect by EWATx procedure (38). Normal estrous cycles were observed in females following PWATx surgeries in the current study, suggesting normal female gonadal function. Whether or not partial function of testis or ovary was affected by E/PWATx procedure is unknown because male and female reproductive status (neither weight of gonads nor reproductive hormone profile) was directly investigated. Thus, variation in glucose tolerance among different surgical groups due to possible disruption of reproductive function could not be excluded. Physiological levels of both testosterone and estrogen have roles in maintaining normal insulin sensitivity (28). Hypogonadism and decreased levels of sex hormones are associated with visceral obesity and insulin resistance in men (20) and women (23), whereas testosterone replacement in middle-aged men (29) or estrogen replacement in postmenopausal women (3) is associated with improved glucose homeostasis and insulin sensitivity. Estrogen-insufficient mice with mutations in the aromatase gene (24) or estrogen receptor (33) develop insulin resistance. Therefore, lack of estrogen in females or lack of testosterone in males results in the development of a metabolic syndrome. If E/PWATx procedure caused hypogonadism and decreased circulating gonadal hormones, deteriorated instead of improved glucose tolerance would be expected to occur.

EWAT is the most frequently studied WAT, and the effect of EWATx on glucose homeostasis has been documented in several studies (5, 16). The contribution of the analogous female gonadal PWAT to glucose homeostasis has not previously been examined. We found that removal of EWAT improved glucose tolerance when mice were fed low-fat chow. Interestingly, removal of PWAT in female mice improved glucose tolerance in both chow- and HFD-fed mice. Thus, although EWAT is the most studied specific fat pad because it is easy to locate and manipulate in male rodents, there is a clear and important role for PWAT that requires further examination and argues for the inclusion of female mice in these studies.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56863, DK-17844 (to S. C. Woods), DK-54080, and DK-73505 (to R. J. Seeley), and National Research Service Award DK-75255 (to H. Shi).


   ACKNOWLEDGMENTS
 
We thank Joyce Sorrell and Kathi Smith for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. J. Seeley, 2170 E. Galbraith Rd., Dept. of Psychiatry, Univ. of Cincinnati, Cincinnati, OH 45237 (e-mail: randy.seeley{at}uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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H. Shi, A. D. Strader, J. E. Sorrell, J. B. Chambers, S. C. Woods, and R. J. Seeley
Sexually different actions of leptin in proopiomelanocortin neurons to regulate glucose homeostasis
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