NIH Public Access Author Manuscript Physiol Behav. Author manuscript; available in PMC 2011 September 1.
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Published in final edited form as: Physiol Behav. 2010 September 1; 101(2): 282–288. doi:10.1016/j.physbeh.2010.05.014.
Transplantation or Removal of Intra-abdominal Adipose Tissue Prevents Age-Induced Glucose Insensitivity Michelle T. Foster1, Haifei Shi3, Randy J. Seeley2, and Stephen C. Woods1 of Psychiatry, Obesity Research Center, University of Cincinnati, Cincinnati, OH 45237 1Department
2Department
of Internal Medicine, Obesity Research Center, University of Cincinnati, Cincinnati,
OH 45237 3Department
of Zoology, Miami University, Oxford, OH 45056
Abstract NIH-PA Author Manuscript NIH-PA Author Manuscript
Increases in intra-abdominal fat, a common feature associated with aging, is an established risk factor for insulin resistance, diabetes and the metabolic syndrome. To examine the direct contribution of intra-abdominal fat in the pathophysiology of insulin resistance we altered fat volume via removal or transplantation in a naturally occurring age-induced moderate model of obesity and insulin resistance. This was accomplished by bilateral removal of epididymal white adipose tissue (Lipx) or transplantation of donor fat into the intra-abdominal side of the peritoneal cavity of 28-wk old rats. Control animals received sham surgery. Glucose tolerance was evaluated at baseline and 4 and 8 wks post-surgery in all groups, and fasting insulin and leptin were additionally measured in 28-wk old rats. In addition, fasted and fed triglyceride, cholesterol and fatty acid concentrations were measured. Before surgery 28-wk old rats weighed more and were glucose intolerant compared with 8-wk old controls. Both Lipx and transplantation significantly prevented age-induced decreases in glucose tolerance, with Lipx causing improvement at 4 wks which declined by 8 wks; and with a significant transplantation improvement at 8 wks only. Lipx significantly increased insulin secretion 15 min after a bolus injection of 0.75 mg/kg dextrose at 4 and 8 wks compared with controls, while transplantation caused a significant (~220%) increase in fasted leptin level at 4 wks only. Taken together, these data suggest that surgical removal or addition of intra-abdominal fat prevents age-induced insulin resistance by different mechanisms and is a suitable model to investigate naturally occurring obesity.
Keywords Lipectomy; Transplantation; Insulin Sensitivity; Glucose Tolerance
© 2010 Elsevier Inc. All rights reserved. To whom correspondence should be addressed: Dr. Michelle T. Foster, 2170 E.Galbraith Road, Department of Psychiatry, University of Cincinnati, Cincinnati, Ohio 45237, 513-558-6590 (Phone), 513-558-5783 (Fax),
[email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Introduction NIH-PA Author Manuscript
Aging in humans and rodents is accompanied by many of the same deleterious metabolic symptoms (8) observed in their respective younger counterparts that are obese. Age-induced obesity, however, is initiated by progressive decreases in metabolic rate, energy expenditure (14) and the thermal response to feeding (34), as well as a tendency toward steatosis (35) and/or impairment in central homeostatic circuits (26), than to intake of high-calorie/ calorically dense foods. Previous research has demonstrated associations among location of fat storage, insulin sensitivity and the concurrent impact on multiple other metabolic complications. For example, insulin resistance and the metabolic syndrome are established consequences of expanding visceral fat mass (23), whereas expansion of subcutaneous fat can be associated with improved glucose tolerance and a lower risk of developing obesityrelated co-morbidities (29,36,37). Age-associated obesity is characterized by increased body fat, primarily visceral fat in humans (10) and rodents (8), and is strongly associated with hepatic and peripheral insulin resistance (5,11,12,15,31,32). In particular, age-induced insulin resistance is a result of progressive increases in fasting and postprandial plasma insulin concentrations, and thus is associated with impaired glucose tolerance and increased risk for type-2 diabetes (T2D) (7,8,33). However, it has not been demonstrated whether abdominal obesity and dyslipidemia are the causes or the result of impaired glucose tolerance and insulin resistance in the obesity that develops with natural aging.
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Although adipose tissue dysregulation has not been directly implicated as a major contributing factor of the metabolic syndrome, evidence demonstrates that a selective reduction in adipose tissue mass improves the metabolic profile. For example, removal of fat via lipectomy (Lipx), specifically intra-abdominal epipdidymal and/or retroperitoneal depots in rodents, reverses insulin resistance and glucose intolerance in obese, aged and young rodents (4,16,22). In humans, visceral omental fat removal improves insulin action (38), whereas removal of non-visceral fat has no effect (23). The altered insulin sensitivity following fat removal may not be dictated by fat removal alone, but may be based in part on some other consequence of Lipx. For example, in both humans and rodents Lipx causes compensatory increases in the mass of non-excised adipose depots. More specifically, liposuction in humans increases body fat in non-excised areas (25,41), as does Lipx in rodents [For review see (28)]. For example, in Siberian hamsters epididymal WAT (EWAT) removal results in increased inguinal WAT (IWAT), peritoneal WAT (PWAT) and dorsosubcutaneous WAT (DWAT) (27).
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Studies have begun to dissect the relative importance of increased visceral fat deposition and adipose depot-specific characteristics via the method of transplantation of adipose tissue. Paradoxically, like Lipx, transplantation of intra-abdominal EWAT or subcutaneous adipose tissue mimicking increased visceral fat is associated with an improved metabolic profile, with some reports indicating that implantation of specific fat depots can elicit cellautonomous distinctive changes of glucose tolerance and insulin sensitivity (20,21,24). Furthermore, reports in which adipocytes were transplanted between lean and genetically obese mice indicate that abnormalities of obese-derived adipose tissue are due to extrinsic as opposed to intrinsic factors (2). It is necessary to note that previous Lipx and transplantation studies in rodents are somewhat limited because there is ambiguity as to which adipose depots in the abdomen are considered visceral. Because EWAT in male rodents and PWAT are in an intra-abdominal location, many researchers have considered EWAT to be a visceral depot. However, because of its drainage into systemic circulation and not into portal circulation to the liver, EWAT should not be considered a visceral depot (19). EWAT also has no human equivalent. Although rodents do have visceral depots similar to those found in humans; i.e., fat deposited in the
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lesser omentum attached to the top edge of the stomach extending to the undersurface of the liver, and in the mesenterium of the intestines, we investigate manipulations of EWAT in the present series of experiments in order to make comparisons to the larger literature. The aim of the present work was to determine the effects of Lipx or transplantation on glucose tolerance in naturally-occurring, age-induced obesity in rats.
Research Design and Methods Animals Adult male 28-wk old (~425 g) and 8-wk old (~250) Long-Evans rats from Harlan (Indianapolis, IN) were individually housed under controlled conditions (12:12 light-dark cycle, 50–60% humidity, and 25° C) with free access to low-fat pelleted chow (Harlan Teklad, Madison, WI; rodent diet; LM485) and water. After 2 weeks acclimation in individual cages, surgeries were performed. Food intake and body mass were measured daily for a week after surgeries, with the day before surgery used as basal body mass. Body mass was also recorded at 4 and 8 wks post surgery. All procedures were approved by the University of Cincinnati Institutional Animal Care and Use Committee. Adipose Lipx, Transplantation and Sham Surgeries
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The rats were anesthetized with isoflurane and injected subcutaneously with buprenex analgesic (300 μg/kg). A mid-ventral abdominal incision was placed between the targeted bilateral pads. The epididymal fat pads (EWAT) were separated from surrounding tissue, tied off, excised (Lipx; n = 4), weighed and placed into the intra-abdominal cavity of the transplant recipient (Transplantation; n= 4). During EWAT removal care was taken not to disrupt major vasculature or reproductive organs. The muscle and skin were sutured with absorbable suture. Roughly 5 g of adipose tissue was removed and subsequently used for transplantation. Sham controls only differed in that the pads were identified but not tied off or excised (28-wk old Controls; n = 4, 8-wk old Controls; n = 8). Animals that received the transplants had a mid-ventral incision in the abdomen; whole donor adipose tissue was attached bilaterally, ~2.5 g to the left side and the remaining ~2.5 g to the right, to the intraabdominal side of the peritoneum at the level of the liver using VETBOND tissue adhesive (3M Center, St. Paul, MN). Glucose Tolerance Test (ipGTT)
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Intraperitoneal (ip) glucose tolerance tests (GTTs) were conducted in all groups before surgery and at 4 and 8 wks post-surgery. Over-night fasted rats (16 h) were moved to the procedure room (0700 h) and after 2 h, baseline blood glucose was obtained in duplicate from the tail vein using a glucometer and glucose strips (FreeStyle, Alameda, CA), and 250 l of blood was collected from the 28-wk old animals into heparin-containing tubes and placed on ice for subsequent insulin and leptin assay. Rats then received a 1.5 mg/kg bolus of 50% dextrose ip. Duplicate blood samples were taken and assessed for glucose after 15, 30, 45, 60 and 120 min, and blood samples from the 28-wk old animals was also collected for insulin assay at 15 min. Leptin and Insulin Concentration Blood from the 28-wk old animals was cold-centrifuged and plasma was frozen at −80° C until insulin and leptin were assessed using ELISA assays (CrystalChem, Inc., Downers Grove, IL).
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Terminal Measurements (Blood Collection and Tissue Harvesting)
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One week after the final ipGTT rats were decapitated after an injection of fatal plus (Vortech Pharmaceutical, Dearborn, MI). Trunk blood from the 28-wk old group was collected in heparinized tubes and stored at 4° C until centrifuged for 10 min at 4° C, and the plasma was stored at −80° C until assayed. Inguinal, epididymal, retroperitoneal and mesenteric WAT (IWAT, EWAT, RWAT and MWAT, respectively) and transplanted adipose tissue were harvested from all groups and weighed to the nearest 0.001 g. Adipose tissue collected for transplant verification was immediately stored in 4% paraformaldehyde until processing for histology. Testes from the 28-wk old group were also weighed in the same manner, to verify testes were not damaged due to Lipx. Quantitative Lipid Assays Commercially available kits were used to measure plasma triglycerides (Randox Laboratories, Crumlin, UK), cholesterol (Thermo Fisher Scientific, Middletown, VA) and non-esterfied free fatty acids (NEFA) (Wako Chemicals, Richmond, VA) as per the manufacturers’ instructions. Fasted lipid profiles were measured from plasma collected during the 8-wk GTT basal sample. A week later fed lipid profiles were measured within the same animals from plasma collected at decapitation following free access to chow. Adipose Tissue Histology
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Histology was performed on WAT according to the methods of Giordano et al. (18) and Bartness et al. (13). Briefly, the WAT pads were washed with 0.015 M PBS (pH = 7.4) and then dehydrated with a series of increasing concentrations of ethanol. Specifically, tissues were incubated into 75% ethanol (60 min), 95% ethanol (2 × 75 min), and 100% ethanol (3 × 60 min). After dehydration, fat pads were incubated in xylene (2 × 60 min). Tissues were then infiltrated with paraffin, a paraplast embedding media (Sigma Chemical), at 60° C overnight and then embedded with fresh paraffin. After dehydration and paraffin infiltration adipose depots size was ~2.5cm in length and ~1cm in width. Each pad was sliced crosssectionally across its width using a rotary microtome (American Optical Instrument, Buffalo, NY). More specifically, three 10 μm samples were collected approximately every 100 μm, immediately placed in a tissue-floating water bath (37° C) and subsequently mounted on glass slides. Slides were left on a slide-warming table (37° C) to air-dry overnight. Of the ~40 slides mounted from each adipose sample five slides were randomly selected (five levels of evaluation), deparaffinized with xylene and hydrated with a series of decreasing concentrations of ethanol and distilled water immediately before being counterstained with hematoxylin (Vector Laboratories). The slides were covered with Histomount (National Diagnostics, Atlanta, GA) after dehydration with a series of increasing concentrations of ethanol and xylene. The final product was visualized using light microscopy. Average cell size was determined using AxiVision, Ziess Imaging Software (Thornwood, NY). Only those cells with the typical fat unilocular ring shape were counted from six random fields within each sample section under 40X magnification. Statistical Analysis Data are expressed as mean ± standard error of the mean (SEM). Comparisons between multiple groups such as lipid profile data, leptin and insulin concentration and individual and total adipose mass were made using one-way between-subjects analysis of variance (ANOVA) (SPSS for Windows, release 11.5.0; SPSS, Chicago, IL). ipGTT (Group × Min design (3 × 6)), area under the curve (AUC) (Week × Group design (3 × 4)) and plasma insulin concentration data (Group × Time design (3 × 2)) were analyzed using two-way ANOVA. Post-hoc tests of individual groups were made using Tukey’s tests. For all experiments, differences among groups were considered statistically significant if P ≤ 0.05.
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Exact probabilities and test values were omitted for simplicity and clarity of presentation of the results.
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Results Body Mass and Terminal Tissue Measurements
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The growth curve/body mass of the 28-wk old EWAT Lipx and transplantation groups were equivalent to sham 28-wk old controls groups during the duration of the study (Figure 1). 8wk old controls, however, weighed significantly less at all measured time points compared with 28-wk old groups ((P ≤ 0.05; Figure 1). At termination, blunt dissection and dissecting scope observation confirmed that the transplanted adipose tissue was viable and revascularized and had a normal looking appearance (Figure 5A). Because transplants were large and from aged donors to aged recipients, small spots of necrosis occurred intermittently on the outside of the transplant (Figure 5A: arrows), these spots were removed after the transplant mass was bilaterally excised, but before weighing. Transplanted tissue weighed non-significantly more than its pre-transplanted weight (Table 1). Bilateral pads in Table 1 are averaged together (EWAT, IWAT and PWAT, respectively). The only significant difference among the 28-wk old experimental groups was decreased EWAT mass due surgical removal, and an increase due to transplantation (P ≤ 0.05; Table 1). MWAT, which is indicative of aged-related obesity, was significantly increased in the 28-wk old control and Lipx group compared to the 8-wk old control, as was IWAT of the Lipx group and EWAT of the 28-wk old control and transplantation group. There was no difference in total dissected adipose tissue mass (IWAT + RWAT + EWAT + endogenous MWAT) among the 28-wk old groups (Table 1), but those of the 8-wk old controls weighed significantly less than those of the 28-wk old controls (P ≤ 0.05; Table 1). In those animals that received Lipx, neither testes mass nor appearance (color or vascularization) was different from that of the respective control group, data not shown. Glucose Tolerance Tests
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All 28-wk old groups were matched according to average body mass (Figure 1) and the area under the curve (AUC) of basal GTT. The latter was significantly higher than that in 8-wk old controls (P ≤ 0.05; Figure 2A inset: In the present and all following figures Transplantation = Trans.). In addition, the 15, 45 and 60 min blood glucose levels of 8-wk old young rats were significantly lower than those of 28 wk groups (P ≤ 0.05; Figure 2A). Overall, this implies that the 28-wk old rats had pre-surgery age-related glucose intolerance. At 4 wks post-surgery there was a significant main effect among groups (P ≤ 0.05; Figure 2B), with Lipx, transplantation and the 8–wk old control groups having decreased blood glucose at all time points compared with 24-wk old controls. More specifically, transplantation or removal of EWAT resulted in a significantly decreased level of glucose after 15 min, matching those of 8-wk old control animals (P ≤ 0.05). Only the Lipx group, however, had a significant decrease in AUC compared with controls (P ≤ 0.05). By 8 wks the main effect among groups (P ≤ 0.05; Figure 2C) was more pronounced, with both Lipx and transplantation resulting in improved glucose tolerance, which was roughly equivalent to or better than that of the 8–wk old control animals. However, glucose levels at specific time points were not different between the 28-wk old control and Lipx groups. In contrast, transplantation caused significant decreases in blood glucose at all time points except 120 min relative to levels in 28-wk old controls (Figure 2C). This persistent diminution resulted in a significant Group × Min interaction (P ≤ 0.05), with the transplantation group having a significantly lower 8 wk AUC compared with both 8- and 28-wk controls and Lipx (P ≤ 0.05; Figure 2C inset), while that of the Lipx group approached significance (P = 0.078). GTT AUC values across weeks demonstrate age-associated changes in glucose tolerance in both 8- and 28-wk old control groups. More specifically, there is a main effect for groups (P
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≤ 0.05) due to higher AUC in 28-wk old controls compared with Lipx, transplantation and 8-wk old controls. There is also a main effect for week with an overall increase in AUC in the control groups over experimental duration (P ≤ 0.05). Lastly, a Group × Week interaction with both 8- and 28-wk old control groups GTT AUC significantly increasing from basal measurement to 8 wks, while Lipx and transplantation either remained consistent or decreased (P ≤ 0.05). Insulin and Leptin Concentrations Before surgery there were no significant differences in basal or 15-min insulin levels among the groups (Figure 3A). By 4 and 8 wks post-surgery, there were significant effects of time and group, and a group × time interaction after glucose injection (Figure 3 B and C). More specifically, although basal values were not different among groups at 4 and 8 wks, the Lipx group had significantly higher insulin than control and transplantation animals at 15 min post glucose injection (P ≤ 0.05). Since pre-surgical (basal) leptin values were quite variable, subsequent data are expressed as a percent of each subject’s basal value (Figure 4). There was no main effect for time indicating that over the course of 8 wks, plasma leptin did not change; however, there was a main effect for group (P ≤ 0.05), and the group × time interaction approached significance (P = 0.071), with the transplantation group having significantly higher leptin at 4 but not 8 wks compared to Lipx and controls (P ≤ 0.05).
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Lipid Profile and Adipose Tissue Histology Terminal non-fasting plasma triglyceride concentration was significantly higher in the transplantation group compared with the Lipx only (P ≤ 0.05; Table 2), but cholesterol and NEFA remained unchanged among the experimental groups. The mean area of transplanted individual adipoctyes was the same as that in the respective endogenous adipose tissue (Figure 5B). Transplanted tissue had normal appearing unilocular rings of fat cells, with intermittent macrophages and vascular cells (Figure 5C).
Discussion
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Human studies indicate that increased intra-abdominal adipose tissue is associated with impaired glucose tolerance, insulin resistance and increased triglyceride secretion (1,9), and intra-abdominal adipose volume is a reliable predictor of diabetes and associated metabolic risks (17,40). However, the mechanisms responsible for the deleterious metabolic effects of intra-abdominal adipose tissue are unknown, and prior research has not clarified whether intra-abdominal obesity is a consequence or contributing factor to insulin resistance and/or diabetes. It is therefore important to develop models to dissect these possibilities experimentally. In the present study, we observed that, in an age-induced model of glucose intolerance in rats fed a low-fat diet, either removal of fat or receiving additional fat via transplantation improved glucose tolerance, although the mechanisms by which the alterations occur may be different. The present results support the findings of previous studies assessing glucose tolerance after transplantation (20,21,24) or Lipx (4,16,22). The present study, however, extends previous research in two ways: 1) utilization of a naturally occurring model of glucose intolerance; and 2) a direct comparison of fat transplantation with Lipx via equivalent fat decreases or increases. In addition, we demonstrate younger animals are more glucose sensitive than aged though both control groups GTT area under the curve increases by ~5,000 from basal to 4wks and does so again from 4wks to 8wks. Therefore, young age does not prevent or restrain a decline in glucose tolerance, because they too are aging and increasing adiposity through-out the study. Consistent with preceding Lipx studies in which glucose tolerance was improved independent of the fat depot removed (4,16,22), removing epididymal fat improved glucose
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tolerance in the present experiment. This occurred despite no change in overall body or adipose tissue mass relative to sham-operated controls. In the present experiment it should be noted that only EWAT was removed whereas other studies removed both EWAT and perinephric WAT (PWAT) (4,16,22). Thus, the total amount of fat removed relative to body mass was significantly less in our model, and leads to the conclusion that improvement of glucose tolerance may be accomplished without removal of multiple or large amounts of intra-abdominal adipose depots.
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A logical conclusion from the Lipx part of the experiment is that the presence of epididymal fat in the body (at least in its natural location) has effects that contribute to increased glucose intolerance as a rat ages. A logical extension of that conclusion might be that having extra epididymal fat would cause a worsening of glucose tolerance. We tested this possibility by taking the epididymal fat removed in the Lipx process and subsequently used it as donor tissue for the transplantation group. Intra-abdominal transplantation of large amounts of fat is a relatively novel animal model which has only been investigated in lean rodents (20,21,24) such that the present study is the first to use a natural model of obesity to investigate changes in glucose homeostasis. Contrary to what might be expected, but consistent with previous reports (20,21,24), when rats received epididymal fat into the abdominal visceral cavity, their glucose tolerance was actually improved. As done in previous studies(24,39), success of transplantation was confirmed histologically via comparison of mean area of transplanted adipocytes with that of endogenous EWAT. It is not clear from prior reports as to which donor depot best improves glucose tolerance when added into the abdominal cavity, with different studies claiming subcutaneous (inguinal; IWAT) (20,39) and others claiming EWAT (24). Those reports differed with regard to the amount of fat transplanted and the location of the transplanted tissue within the recipient animal. Some placed the transplant onto the peritoneal surface of the anterior abdominal wall (20,24) and others placed it deeper within the intra-abdominal cavity lodged between endogenous adipose tissue folds (39). In addition, the time(s) post-surgery when assessments of glucose tolerance were made may also be a contributing factor in whether or not EWAT transplantation improves glucose tolerance, with 8 wks resulting in a significant improvement in one report (24), as was also observed in our study, or assessment at 12 or 13 wks (20,39) resulting in no difference. Thus, it is possible that EWAT transplantation may have a transient beneficial effect on glucose tolerance.
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Assessment of fasting insulin and leptin suggest that the improved glucose tolerance may transpire by different mechanisms depending upon whether intra-abdominal fat was removed or added. Transplantation of EWAT to the intra-abdominal cavity did not alter insulin secretion compared with controls at any time point, which is consistent with some (39), but not all previous studies (24). Basal insulin was also unaltered following Lipx, but subsequent to a bolus glucose injection, Lipx rats had increased insulin at 4 and 8 wks after surgery. In young lean or diabetic animals, intra-abdominal fat removal decreased basal insulin by ~50% and reduced the insulin infusion rate needed to maintain steady glucose levels during a hyperinsulinemic-euglycemic clamp (4,16). In contrast, yet consistent with our data, basal insulin levels following Lipx in obese models remained unchanged (16,22), implying that improved glucose tolerance need not be secondary to altered basal insulin. Improved glucose removal in the Lipx group may, however, be a consequence of increases in glucose-stimulated insulin secretion that we observed at both 4 and 8 wks. Increased glucose-stimulated insulin secretion is typically associated with decreased insulin sensitivity, poorer glucose tolerance, increased fat deposition and hypertriglyceridemia (3,6), but the only one of these present in our model is the possibility of decreased insulin sensitivity. Further studies are needed to investigate Lipx-induced duration of elevated insulin concentration, since only a single glucose-induce measure of insulin concentration
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was collected, and if this alteration in insulin secretion is a contributing factor of Lipxinduced improvements in glucose clearance.
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Consistent with no change of total fat, removal of EWAT in the present study did not alter leptin concentrations, and this is consistent with a previous report (30). There was an apparent transient increase of leptin at 4 wks post-transplantation, but it was gone by 8 wks, consistent with reports that measured leptin after 10 (24) or 12 wks (39). This study is the first to reveal a transplantation-induced transient increase in leptin. Collectively, the present results indicate that surgical removal or addition of intra-abdominal fat, EWAT, prevents age-induced insulin resistance, with Lipx enhancing insulin secretion and glucose clearance, and transplantation improving insulin sensitivity. Ultimately understanding the complex interactions among cell-autonomous fat depots, liver, age, vasculature and resulting circulating hormones is a new area that can yield important information about development and prevention of insulin resistance and type-2 diabetes.
Acknowledgments This research was supported by National Institutes of Health
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References
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1. Adiels M, Taskinen MR, Packard C, Caslake MJ, Soro-Paavonen A, Westerbacka J, Vehkavaara S, Hakkinen A, Olofsson SO, Yki-Jarvinen H, Boren J. Overproduction of large VLDL particles is driven by increased liver fat content in man. Diabetologia 2006;49:755–765. [PubMed: 16463046] 2. Ashwell M. The use of the adipose tissue transplantation technique to demonstrate that abnormalities in the adipose tissue metabolism of genetically obese mice are due to extrinsic rather than intrinsic factors. Int J Obes 1985;9(Suppl 1):77–82. [PubMed: 3905651] 3. Bard JM, Charles MA, Juhan-Vague I, Vague P, Andre P, Safar M, Fruchart JC, Eschwege E. Accumulation of triglyceride-rich lipoprotein in subjects with abdominal obesity: the biguanides and the prevention of the risk of obesity (BIGPRO) 1 study. Arterioscler Thromb Vasc Biol 2001;21:407–414. [PubMed: 11231921] 4. Barzilai N, She L, Liu BQ, Vuguin P, Cohen P, Wang J, Rossetti L. Surgical removal of visceral fat reverses hepatic insulin resistance. Diabetes 1999;48:94–98. [PubMed: 9892227] 5. Carey DG, Jenkins AB, Campbell LV, Freund J, Chisholm DJ. Abdominal fat and insulin resistance in normal and overweight women: Direct measurements reveal a strong relationship in subjects at both low and high risk of NIDDM. Diabetes 1996;45:633–638. [PubMed: 8621015] 6. Chan DC, Watts GF, Barrett PH, Mamo JC, Redgrave TG. Markers of triglyceride-rich lipoprotein remnant metabolism in visceral obesity. Clin Chem 2002;48:278–283. [PubMed: 11805008] 7. Chang AM, Halter JB. Aging and insulin secretion. Am J Physiol Endocrinol Metab 2003;284:E7– 12. [PubMed: 12485807] 8. Das M, Gabriely I, Barzilai N. Caloric restriction, body fat and ageing in experimental models. Obes Rev 2004;5:13–19. [PubMed: 14969503] 9. Despres JP, Lemieux S, Lamarche B, Prud’homme D, Moorjani S, Brun LD, Gagne C, Lupien PJ. The insulin resistance-dyslipidemic syndrome: contribution of visceral obesity and therapeutic implications. Int J Obes Relat Metab Disord 1995;19(Suppl 1):S76–86. [PubMed: 7550542] 10. Enzi G, Gasparo M, Biondetti PR, Fiore D, Semisa M, Zurlo F. Subcutaneous and visceral fat distribution according to sex, age, and overweight, evaluated by computed tomography. Am J Clin Nutr 1986;44:739–746. [PubMed: 3788827] 11. Facchini FS, Hua N, Abbasi F, Reaven GM. Insulin resistance as a predictor of age-related diseases. J Clin Endocrinol Metab 2001;86:3574–3578. [PubMed: 11502781] 12. Ferrannini E, Vichi S, Beck-Nielsen H, Laakso M, Paolisso G, Smith U, European Group for the Study of Insulin Resistance (EGIR). Insulin action and age. Diabetes 1996;45:947–953. [PubMed: 8666147]
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13. Foster MT, Bartness TJ. Sympathetic but not sensory denervation stimulates white adipocyte proliferation. Am J Physiol Regul Integr Comp Physiol 2006;291:R1630–1637. [PubMed: 16887921] 14. Fukagawa NK, Bandini LG, Young JB. Effect of age on body composition and resting metabolic rate. Am J Physiol 1990;259:E233–238. [PubMed: 2382714] 15. Gabriely I, Barzilai N. Surgical removal of visceral adipose tissue: effects on insulin action. Curr Diab Rep 2003;3:201–206. [PubMed: 12762966] 16. Gabriely I, Ma XH, Yang XM, Atzmon G, Rajala MW, Berg AH, Scherer P, Rossetti L, Barzilai N. Removal of visceral fat prevents insulin resistance and glucose intolerance of aging: an adipokine-mediated process? Diabetes 2002;51:2951–2958. [PubMed: 12351432] 17. Gastaldelli A, Miyazaki Y, Pettiti M, Matsuda M, Mahankali S, Santini E, DeFronzo RA, Ferrannini E. Metabolic effects of visceral fat accumulation in type 2 diabetes. J Clin Endocrinol Metab 2002;87:5098–5103. [PubMed: 12414878] 18. Giordano A, Morroni M, Santone G, Marchesi GF, Cinti S. Tyrosine hydroxylase, neuropeptide Y, substance P, calcitonin gene-related peptide and vasoactive intestinal peptide in nerves of rat periovarian adipose tissue: an immunohistochemical and ultrastructural investigation. J Neurocytol 1996;25:125–136. [PubMed: 8699194] 19. Harris RB, Leibel RL. Location, location, location. Cell Metab 2008;7:359–361. [PubMed: 18460325] 20. Hocking SL, Chisholm DJ, James DE. Studies of regional adipose transplantation reveal a unique and beneficial interaction between subcutaneous adipose tissue and the intra-abdominal compartment. Diabetologia 2008;51:900–902. [PubMed: 18340430] 21. Kahn BB, Flier JS. Obesity and insulin resistance. J Clin Invest 2000;106:473–481. [PubMed: 10953022] 22. Kim YW, Kim JY, Lee SK. Surgical removal of visceral fat decreases plasma free fatty acid and increases insulin sensitivity on liver and peripheral tissue in monosodium glutamate (MSG)-obese rats. J Korean Med Sci 1999;14:539–545. [PubMed: 10576150] 23. Klein S, Fontana L, Young VL, Coggan AR, Kilo C, Patterson BW, Mohammed BS. Absence of an effect of liposuction on insulin action and risk factors for coronary heart disease. N Engl J Med 2004;350:2549–2557. [PubMed: 15201411] 24. Konrad D, Rudich A, Schoenle EJ. Improved glucose tolerance in mice receiving intraperitoneal transplantation of normal fat tissue. Diabetologia 2007;50:833–839. [PubMed: 17334653] 25. Lambert EV, Hudson DA, Bloch CE, Koeslag JH. Metabolic response to localized surgical fat removal in nonobese women. Aesthetic Plast Surg 1991;15:105–110. [PubMed: 2035358] 26. Matsumoto AM, Marck BT, Gruenewald DA, Wolden-Hanson T, Naai MA. Aging and the neuroendocrine regulation of reproduction and body weight. Exp Gerontol 2000;35:1251–1265. [PubMed: 11113606] 27. Mauer MM, Bartness TJ. Body fat regulation after partial lipectomy in Siberian hamsters is photoperiod dependent and fat pad specific. Am J Physiol 1994;266:R870–878. [PubMed: 8160883] 28. Mauer MM, Harris RB, Bartness TJ. The regulation of total body fat: lessons learned from lipectomy studies. Neurosci Biobehav Rev 2001;25:15–28. [PubMed: 11166075] 29. Misra A, Garg A, Abate N, Peshock RM, Stray-Gundersen J, Grundy SM. Relationship of anterior and posterior subcutaneous abdominal fat to insulin sensitivity in nondiabetic men. Obes Res 1997;5:93–99. [PubMed: 9112243] 30. Nogalska A, Stelmanska E, Sledzinski T, Swierczynski J. Surgical removal of perirenal and epididymal adipose tissue decreases serum leptin concentration and increases lipogenic enzyme activities in remnant adipose tissue of old rats. Gerontology 2009;55:224–228. [PubMed: 19088460] 31. O’Shaughnessy IM, Myers TJ, Stepniakowski K, Nazzaro P, Kelly TM, Hoffmann RG, Egan BM, Kissebah AH. Glucose metabolism in abdominally obese hypertensive and normotensive subjects. Hypertension 1995;26:186–192. [PubMed: 7607722] 32. Peiris AN, Struve MF, Mueller RA, Lee MB, Kissebah AH. Glucose metabolism in obesity: influence of body fat distribution. J Clin Endocrinol Metab 1988;67:760–767. [PubMed: 3047162]
Physiol Behav. Author manuscript; available in PMC 2011 September 1.
Foster et al.
Page 10
NIH-PA Author Manuscript NIH-PA Author Manuscript
33. Reaven GM, Reaven EP. Age, glucose intolerance, and non-insulin-dependent diabetes mellitus. J Am Geriatr Soc 1985;33:286–290. [PubMed: 3886767] 34. Schwartz RS, Jaeger LF, Veith RC. The thermic effect of feeding in older men: the importance of the sympathetic nervous system. Metabolism 1990;39:733–737. [PubMed: 2195295] 35. Sepe A, Tchkonia T, Thomou T, Zamboni M, Kirkland JL. Aging and Regional Differences in Fat Cell Progenitors - A Mini-Review. Gerontology. 36. Snijder MB, Dekker JM, Visser M, Bouter LM, Stehouwer CD, Kostense PJ, Yudkin JS, Heine RJ, Nijpels G, Seidell JC. Associations of hip and thigh circumferences independent of waist circumference with the incidence of type 2 diabetes: the Hoorn Study. Am J Clin Nutr 2003;77:1192–1197. [PubMed: 12716671] 37. Tanko LB, Bagger YZ, Alexandersen P, Larsen PJ, Christiansen C. Peripheral adiposity exhibits an independent dominant antiatherogenic effect in elderly women. Circulation 2003;107:1626–1631. [PubMed: 12668497] 38. Thorne A, Lonnqvist F, Apelman J, Hellers G, Arner P. A pilot study of long-term effects of a novel obesity treatment: omentectomy in connection with adjustable gastric banding. Int J Obes Relat Metab Disord 2002;26:193–199. [PubMed: 11850750] 39. Tran TT, Yamamoto Y, Gesta S, Kahn CR. Beneficial effects of subcutaneous fat transplantation on metabolism. Cell Metab 2008;7:410–420. [PubMed: 18460332] 40. Vega GL, Adams-Huet B, Peshock R, Willett D, Shah B, Grundy SM. Influence of body fat content and distribution on variation in metabolic risk. J Clin Endocrinol Metab 2006;91:4459– 4466. [PubMed: 16926254] 41. Yost TJ, Rodgers CM, Eckel RH. Suction lipectomy: outcome relates to region-specific lipoprotein lipase activity and interval weight change. Plast Reconstr Surg 1993;92:1101–1108. discussion 1109-1111. [PubMed: 8234508]
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NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 1.
Basal, 4 and 8 wk absolute body mass (Mean ± SEM. (g)) of 8 and 28 wk groups. * = P ≤ 0.05 for 8 wk controls vs. 28 wk controls, Lipx and Transplant group.
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Figure 2.
8 and 28 wk groups glucose tolerance at 0, 15, 30, 45, 60 and 120 min post-glucose injection (ip.) at basal (A), 4 (B) and 8 (C) wks with area under the curve (AUC) of respective wk (A, B and C Inset) (Mean ± SEM (mg/dl)). Number in wks on graphs indicates the age groups started at. A.) All 28 wk groups basal glucose tolerance was higher than 8 wk controls at 15, 45 and 60 mins (* = P ≤ 0.05); 8 wk controls had decreased AUC. B.) 4 wk - Lipx and transplantation decreased glucose peak at 15 min to 8 wk control levels (* = P ≤ 0.05 vs. 28 wk controls); Lipx decreased AUC. C.) 8 wk – 28 wk controls had elevated glucose at 15, 30, 45 and 60 mins compared with 8 wk controls (* = P ≤ 0.05); 28 wk controls had higher AUC. Transplantation decreased glucose peak at 15 (** = P ≤ 0.05 vs. 8 and 28 wk controls and Lipx), 30, 45, 60 and 120 mins (*= P ≤ 0.05 vs. 28 wk controls); Transplantation decreased AUC. For all insets non-shared letters (a,b,c) indicate significant difference (P ≤ 0.05).
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NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 3.
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Plasma insulin concentration at 0 and 15 mins post-glucose injection (ip.) in 28 wk groups only at basal (A), 4 (B) and 8 (C) wks (Mean ± SEM (ng/ml)). A.) Basal insulin. B.) 4 and C.) 8 wks – Lipx increased insulin at 15 mins. Non-shared letters (a,b,c) indicate significant difference (P ≤ 0.05).
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Figure 4.
Percent plasma leptin concentration relative to basal in 28 wk group only. Transplantation increased leptin at 4 wks only. Non-shared letters (a,b) indicate significant difference (P ≤ 0.05).
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Figure 5.
Transplant verification. A.) Blunt dissection: Representative picture of transplanted adipose tissue that was viable, revascularized and a normal looking appearance. Arrows indicated spots of necrosis that occurred intermittently. B.) Adipocyte cell area (μm2): Left and right transplanted adipose tissue cell area is not different than control. C.) Adipose tissue histology: Left panel = control adipose tissue, middle = left transplanted tissue, and right = right transplanted tissue.
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Table 1
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Absolute mass of testes and removed, implanted or recovered transplant as well as individual and total dissected adipose tissue (Mean ± SEM. (g)) of all groups. Bilateral adipose depots, respectively EWAT, IWAT and PWAT, are summed together. Non-shared letters (a,b,c) indicate significant difference (P ≤ 0.05) 28wk Control
28wk Trans
28wk Lipx
8wk Control
Transplanted Or Removed Fat
N/A
5.14± 0.49
5.14± 0.49
N/A
Recovered Transplant
N/A
5.64±0.87
N/A
N/A
EWAT
10.74±1.17a
9.22± 0.32a
3.57±0.38b
6.76±0.22c
MWAT
10.79± 0.92a
9.71±0.96a,b
11.59±1.37a
8.58±0.46b
IWAT
13.12±1.73a,b
12.57±2.19a,b
15.24±3.lla
9.14±0.30b
PWAT
11.17±1.88
10.16±1.49
11.04±1.53
7.84±0.88
Total Dissected Fat
45.83±5.50a
47.30±4.13a
41.43±6.00a
32.18±1.38b
1.788±0.0662
1.686±0.067
1.661±0.105
N/A
Testes
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Table 2
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28 wk groups fed and fasted triglyceride, cholesterol and NEFA concentrations. Non-shared letters (a,b) indicate significant difference (P ≤ 0.05) Control Triglycerides mg/dl Fed
Fasted
147.893±33.43a,b
Transplantation 191.74± 12.12a
Lips 127.91± 20.07b
Cholesterol mg/dl
85.62± 10.64
88.03± 6.57
78.75±10.02
NEFA mmol/L
0.22± 0.023
0.21± 0.023
0.27± 0.041
Triglycerides mg/dl
122.75±11.85
111.49± 6.84
97.467± 9.04
Cholesterol mg/dl
78.92± 11.18
75.30± 4.50
64.86±4.75
NEFA mmol/L
1.97± 0.095
1.91 ± 0.180
2.16± 0.141
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