Improved glucose tolerance in mice receiving intraperitoneal transplantation of normal fat tissue
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- Konrad, D., Rudich, A. & Schoenle, E.J. Diabetologia (2007) 50: 833. doi:10.1007/s00125-007-0596-1
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The association between increased (visceral) fat mass, insulin resistance and type 2 diabetes mellitus is well known. Yet, it is unclear whether the mere increase in intra-abdominal fat mass, or rather functional alterations in fat tissue in obesity contribute to the development of insulin resistance in obese patients. Here we attempted to isolate the metabolic effect of increased fat mass by fat tissue transplantation.
Epididymal fat pads were removed from male C57Bl6/J mice and transplanted intraperitoneally into male littermates (recipients), increasing the combined perigonadal fat mass by 50% (p < 0.005). At 4 and 8 weeks post-transplantation, glucose and insulin tolerance tests were performed, and insulin, NEFA and adipokines measured.
Circulating levels of NEFA, adiponectin and leptin were not significantly different between transplanted and sham-operated control mice, while results of the postprandial insulin tolerance test were similar between the two groups. In contrast, under fasting conditions, the mere increase in intra-abdominal fat mass resulted in decreased plasma glucose levels (6.9 ± 0.4 vs 8.1 ± 0.3 mmol/l, p = 0.03) and a ∼20% lower AUC in the glucose tolerance test (p = 0.02) in transplanted mice. Homeostasis model assessment of insulin resistance (HOMA-IR) was 4.1 ± 0.4 in transplanted mice (vs 6.2 ± 0.7 in sham-operated controls) (p = 0.02), suggesting improved insulin sensitivity. Linear regression modelling revealed that while total body weight positively correlated, as expected, with HOMA-IR (β: 0.728, p = 0.006), higher transplanted fat mass correlated with lower HOMA-IR (β: −0.505, p = 0.031).
Increasing intra-abdominal fat mass by transplantation of fat from normal mice improved, rather than impaired, fasting glucose tolerance and insulin sensitivity, achieving an effect opposite to the expected metabolic consequence of increased visceral fat in obesity.
KeywordsFat transplantation Glucose homeostasis Insulin resistance Insulin sensitivity Obesity Visceral adiposity
homeostasis model assessment of insulin resistance
intraperitoneal glucose tolerance test
intraperitoneal insulin tolerance test
Obesity is caused by energy intake in excess of energy expenditure, and is associated with an increased number and/or size of white adipocytes, which store the excessive energy in the form of triacylglycerol. The association between increased fat mass, insulin resistance and type 2 diabetes mellitus is well known. Yet, regional body fat distribution may influence the occurrence of insulin resistance associated with obesity. Increased accumulation of visceral (intra-abdominal) fat seems to be particularly associated with an increased risk of insulin resistance and type 2 diabetes. Two major lines of observations suggest a causative role for intra-abdominal fat in this association. First, subjects with normal body weight, but with increased visceral adiposity (e.g. elderly people or patients with certain forms of lipodystrophy) are prone to develop insulin resistance [1, 2]. Second, removal of intra-abdominal fat improved insulin sensitivity [3, 4], whereas the removal of subcutaneous fat tissue by liposuction in obese humans did not improve insulin action . Thus, the pattern of body fat distribution, especially abdominal fat accumulation, determines insulin sensitivity.
One remaining question to define in this process is the relative contributions of: (1) the mere increase in intra-abdominal fat mass; and (2) the role of alterations in fat tissue biology that accompany obesity. Increase in fat mass as such has been suggested to increase the delivery of non-esterified fatty acids through the portal circulation, resulting in increased hepatic and muscular triacylglycerol accumulation . Similarly, altered adipose tissue biology can change inter-organ cross-talk mechanisms through alterations in its metabolic and/or endocrine function. For example, a recent study demonstrated that the degree of macrophage infiltration into omental fat of obese patients correlated with histopathological findings in liver biopsies .
To investigate the isolated role of a mere increase in intra-abdominal fat in mice, we used intra-abdominal fat transplantation to increase the intra-abdominal mass of ‘normal’ fat. We reasoned that if the mere increase in fat mass is the predominant process occurring in obesity, increasing intra-abdominal fat mass by surgical transplantation of fat tissue from a normal, non-obese littermate would induce metabolic consequences in the recipient mice that resemble those occurring in obesity. If, on the other hand, the predominating effect is obesity-induced alterations in the endocrine function of fat tissue, the addition of such fat mass would not impair metabolic regulation.
Materials and methods
Male C57BL6JOlaHsd mice were purchased from Harlan Netherlands (Horst, the Netherlands). All mice were housed in a pathogen-free environment on a 12-h light-dark cycle, with free access to standard rodent diet (Provimi Kliba, Kaiseraugst, Switzerland). All protocols conformed to the Swiss animal protection laws and were approved by the Cantonal Veterinary Office in Zurich, Switzerland.
Transplantation procedure was performed at 6 weeks of age. Mice were anaesthetised with isoflurane (Abbott, Baar, Switzerland). Both epididymal fat pads were removed from the donor mouse, rinsed with 0.9% saline and thereafter stitched to the visceral side of the peritoneum of the recipient mouse using Vicryl 5.0 (Johnson-Johnson, Spreitenbach, Switzerland). Except for a small piece of fat tissue fixed in 4% buffered formalin, the entire two fat pads were weighed and then transplanted. Sham-operated control mice received the same treatment, but instead of the fat pad transplantation, an artificial suture was performed with Vicryl. Subcutaneous injection of buprenorphine every 12 h for 2 days was used for analgesia. In order to prevent transplant rejection, donor and recipient mice were littermates.
Glucose and insulin tolerance tests
For fasting intraperitoneal glucose tolerance test (ipGTT), either prolonged fasting (overnight, i.e. 12 h food withdrawal) or 7 h food withdrawal were used, as indicated. For intraperitoneal insulin tolerance test (ipITT) in the post-absorptive state, mice were fed and then fasted for 3 h. Either glucose (2 g/kg body weight) or human normal insulin (0.75 U/kg body weight) were injected intraperitoneally and blood was collected from the tail vein at different time points, as indicated . Plasma glucose was measured using a glucose meter (Ascensia Contour; Bayer, Zurich, Switzerland).
Determination of plasma insulin, adipokines and NEFA levels
Plasma was collected from the tail vein after 7 h of fasting. Insulin was determined using an ELISA kit (Ultra Sensitive Rat Insulin ELISA; Crystal Chem, Downers Grove, IL, USA). NEFA levels were measured using the ACS-ACOD-MEHA method (Wako Chemicals GmbH, Neuss, Germany). Plasma leptin and adiponectin were determined with commercially available mouse endocrine and single plex adiponectin kits (MENDO-75K and MADPK-71K-ADPN; Linco Research, distributed by Labodia, Yens, Switzerland) that are capable of simultaneously measuring different adipokines in mouse serum or plasma. The two kits are based on Luminex xMAP technology (Linco Research, St Charles, MI, USA) and use microsphere bead sets that are uniquely labelled with a mixture of two fluorescent dyes. The captured antibodies specific for each analyte are covalently coupled to individual bead sets. At the time of the assay, a mixture of beads is incubated overnight at 4°C with 10 μl of standards or mouse plasma samples in a 96-well filter-bottom plate. On the next day, the beads are washed, and biotinylated detection antibody cocktail is added and incubated for 30 min at room temperature, followed by the addition of streptavidin-phycoerythrin and incubation for another 30 min. After a final wash, the resuspended beads are read on a Luminex 100 reader (Bio-Rad Laboratories, Hercules, CA, USA) and the concentration of each analyte in the samples to be tested is determined on the basis of individual standard curves.
Determination of homeostasis model assessment of insulin resistance
The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using glucose and insulin determinations obtained after 7 h of food withdrawal, using the following formula: fasting glucose (mmol/l)×fasting insulin (mU/ml)/22.5 [9, 10].
Epididymal fat tissues were fixed in 4% buffered formalin and imbedded in paraffin. Sections were obtained either at the time of transplantation or 8 weeks after transplantation and stained with haematoxylin-eosin. For each mouse at least four fields (representing a total of 350 to 550 adipocytes) were analysed. Images were analysed using ImageJ software for quantification (National Institutes of Health, Bethesda, MD, USA).
Statistical analyses of the obtained data were performed using Student’s t test or ANOVA (Fisher’s multiple comparisons test), as indicated. We performed Pearson correlations between the continuous variables and estimated the independent effect of fat transplantation by further adjustment for total body weight using a linear regression model (SPSS package software, version 14).
Intraperitoneal fat transplantation increases intra-abdominal fat mass without significantly altering circulating adipokine levels
Total body weight, perigonadal fat pad weight and circulating plasma levels of adipokines and NEFA
Body weight (g)
28.0 ± 0.7
27.4 ± 0.9
Perigonadal depots, total (mg)
365 ± 22
508 ± 23
Endogenous fat pad (mg)
365 ± 22
320 ± 18
Transplanted fat pad (mg)
188 ± 9
13.2 ± 1.0
11.1 ± 0.8
1,330 ± 180
1,075 ± 71
2,214 ± 407
1,912 ± 421
1.14 ± 0.08
1.01 ± 0.04
Recipient mice with transplantation-mediated increase in intra-abdominal fat mass exhibit improved glucose tolerance in the fasting state
It is unclear whether increased intra-abdominal fat mass as such or altered fat tissue biology contributes to the metabolic derangement associated with obesity. The first notion is supported by findings that intra-abdominal fat mass reduction improves insulin sensitivity in rats , whereas implantation of cultured adipocytes into the peritoneal cavity of mice induces insulin resistance . Here we used intra-abdominal fat tissue transplantation to increase fat mass. Surprisingly, lean recipients of epididymal fat transplant from lean littermates exhibited improved fasting glucose tolerance and increased insulin sensitivity. Thus, increasing intra-abdominal fat mass by transplantation of normal adipose tissue did not mimic the effects of increased fat mass in obesity.
In this model, fat mass of transplanted fat tissue was inversely correlated with HOMA-IR, independently of the expected positive association between total body weight and HOMA-IR. Histological examination of the transplanted adipose tissue revealed that after 10 weeks of observation (transplantation to scarification) adipocyte cell size increased significantly in endogenous fat pads, whereas it remained unchanged in the transplanted fat pads (Fig. 2b). Insulin action was previously shown to be dependent on fat cell size: large adipocytes correlate well with insulin resistance [12, 13, 14] and show decreased insulin-stimulated glucose uptake . Interestingly, the transplanted fat depot exhibited features reminiscent of those induced in adipose tissue by thiazolidinediones. These drugs were shown to increase cellularity in adipose tissue by promoting fat cell differentiation and inducing apoptosis of large adipocytes, resulting in smaller, more insulin-sensitive adipocytes that secrete higher levels of adiponectin [16, 17]. It is thus possible that the insulin-sensitising effects of the transplant are secondary to similar alterations in the transplanted adipose tissue. We therefore assessed whether alterations in circulating levels of secretory products of fat tissue mediated the improved glucose tolerance and increased insulin sensitivity in mice receiving fat transplantation. However, we did not observe any significant increases in the circulating levels of potential insulin-sensitising adipokines like leptin or adiponectin  or any significant decreases in factors associated with reduced insulin sensitivity (resistin and NEFA, Table 1).
Although potentially informative, such analysis is limited in at least two major aspects. First, we measured total adiponectin levels, but recent studies suggest that the circulating high-molecular-mass complex of adiponectin correlates better with insulin sensitivity than total serum adiponectin levels [19, 20]. Second, with regard to the unaltered circulating levels of adipokines and NEFA observed by us, it should be noted that venous drainage of intra-abdominal fat is to the portal system. It therefore remains possible that intra-portal levels of secreted products were affected, thereby predominantly influencing liver glucose homeostasis. Such a scenario, in which the greatest effect of intra-abdominal fat tissue transplantation is seen in the liver, is also supported by the fact that the insulin-sensitising effects of intra-abdominal fat transplantation was largely observed in the fasting state, conditions in which glycaemia is largely controlled by hepatic glucose production and insulin sensitivity.
In conclusion, our findings suggest that in addition to increased fat mass, altered adipose tissue function is required to reproduce obesity-related metabolic changes such as insulin resistance. These changes may include impaired metabolic function of adipocytes, changes in cellular composition of adipose tissue (e.g. macrophage infiltration)  and/or alterations in the secretory function of adipose tissue (inflammatory cytokines, adipokines, NEFA). The second possible conclusion is that augmentation of normally functioning adipose tissue appears to result in a new metabolic steady state with increased insulin sensitivity. Such compensation and adaptation mechanisms may be unable to counterbalance larger degrees of fat tissue expansion. Yet, the results suggest that multiple factors in the cross-talk mechanisms between different insulin-sensitive tissues need to be affected in order to impair glucose homeostasis in obesity. The notion that adipose tissue function (rather than mass) and its inter-organ cross-talk are key determinants of obesity-induced metabolic derangement further defines this disorder as an endocrinopathy, and adipose tissue as an endocrine organ.
This work was supported by grants from the Medical Faculty of the University of Zurich and the Swiss National Science Foundation grant no. 310000-112275 (to D. Konrad). We would like to greatly acknowledge I. Shai from the S. Daniel Abraham International Center for Health and Nutrition, Ben-Gurion University for valuable input and guidance in the statistical analysis of our data and M. Arras for her expert veterinary advice.
Duality of interest
No conflict of interest existed for any of the authors.