Body Weight, Body Composition, and Energy Balance
Fat transplantations were performed using subcutaneous (SQ) flank fat and visceral (VIS) epididymal fat of wild-type C57BL/6 donor mice (in cohort 1) or C57BL/6 donor mice transgenic for green fluorescent protein (GFP) expressed on the β-actin promoter (in cohort 2), and placing them into either the dorsal subcutaneous area or intra-abdominal area of 12-week-old wild-type C57BL/6 recipient mice (). During the 12 weeks after surgery, mice in the sham group had steady body weight gain in cohorts 1 and 2. In mice in which visceral fat was added to the visceral cavity (VIS-VIS group), total body weight gained at the end of the study was 108% of that in the sham group in cohort 1 and 88% of that in the sham group in 2 but was not significantly different from both sham groups (). In the group with visceral fat transplanted to the SQ area (VIS-SQ group), body weight was not significantly different from the sham group and was not further studied. In contrast, when SQ fat was transplanted to the SQ area (SQ-SQ group), there was a significant slower increase in body weight, and by 12 weeks, this group had 85% and 84% of the weight gained by the sham group in cohorts 1 and 2 respectively. More striking was the effect of adding SQ fat to the VIS cavity (SQ-VIS group), which had the greatest impact on reducing the rate of weight gain. Thus, by the end of the 12-week study period, mice in the SQ-VIS group had gained on average 63% of the amount gained by the sham group in cohort 1 and 59% of the weight gained in the sham group in cohort 2 (p<0.05). Thus, increasing the amount of SQ fat by transplantation resulted in decreased subsequent weight gain, and this effect was greatest when the SQ fat was added to the VIS cavity. These differences in weight gain between the sham and transplantation groups were due primarily to differences in fat mass as measured by DEXA scan. Thus, total fat mass was similar among the sham, VIS-VIS, and VIS-SQ groups, was decreased by 18% in the SQ-SQ group (p<0.05), and reduced by about 54% in the SQ-VIS group (p<0.01) at the end of the study (). Note that this pattern was similar to that of decreasing body weight in the same groups. Percent body fat, i.e. fat mass divided by total body weight, showed a similar decreasing pattern among the groups, with highest percent body fat in the sham, VIS-VIS, and VIS-SQ, and SQ-SQ groups, and a significant 46% reduction in the SQ-VIS cavity group (p<0.01) (). Absolute lean body mass was significantly higher in the SQ-VIS group than the sham group at 6 and 8 weeks and tended to be higher at 12 weeks (), but in relation to body weight, percent lean mass was highest in the SQ-VIS group in comparison to the sham group (). The difference in body weight gain was not due to differences in food intake. Food intake measured at 10 weeks after transplantation by the CLAMS technique was not significantly different among groups (3.9±0.3, 3.9±0.1, 4.3±0.2, 4.2±0.2 g/day for the sham, VIS-VIS, SQ-SQ, and SQ-VIS groups respectively in cohort 2). The difference in body weight was also not due to levels of energy expenditure because these levels were not significantly different among the sham and all transplantation groups during the light and dark cycles (). Simple regression analyses indicate that neither lean mass nor fat mass correlated with total energy expenditure (r2=0.0002 and r2=0.003 respectively) (StatView, SAS Institute Inc., version 5.0.1). Thus, lean mass and fat mass did not have an independent impact on energy expenditure. Respiratory quotient (RQ) is a ratio between the carbon dioxide production and the oxygen consumption, which is an indicator of the relative level of carbohydrate and fat oxidation in the whole body. RQ was significantly higher in the SQ-to-VIS group than in the sham group by 5.9% during the light cycle (p<0.05), by 7.0% during the dark cycle (p<0.05)(), and almost all 30 minute-intervals during the 24-hour measurement (), indicating a higher proportion of carbohydrate to fat metabolism in the SQ- to-VIS group. Otherwise, activity level, heat production, and water intake as measured by the CLAMS method were not significantly different among the sham and all the transplanted fat groups (data not shown). Overall, the SQ-VIS group had significantly improved metabolism in terms of decreased body weight, percent body fat, and increased percent lean mass, and this was associated with an increased proportion of carbohydrate to fat metabolism without significant changes in total energy expenditure or heat production.
Figure 1 Schematic of Fat Transplantation Groups. Visceral (VIS; epididymal) fat or subcutaneous (SQ; flank) fat from donor mice expressing whole-body GFP was transplanted into the visceral or subcutaneous area of wild-type C57BL/6 host mice. The sham group had (more ...)
Figure 2 Body Weight, Body Composition, Energy Expenditure and Respiratory Quotient After Fat Transplantation. (A, B) In cohorts 1 and 2, body weight gained was similar or higher after VIS fat transplantations, but lower after SQ-SQ fat transplantation and lowest (more ...)
Plasma Levels, Glucose and Insulin Tolerance Tests (GTT, ITT)
At nine weeks after transplantation, the levels of plasma glucose, insulin, leptin and adiponectin were assessed, and these showed a similar pattern to that of body weight and fat mass. Basal plasma glucose levels were similar among the sham, VIS-VIS, and VIS-SQ groups at 145–150 mg/dl, were slightly but significantly decreased (~6%) in the SQ-SQ group (p<0.05), and was even more significantly decreased in these lean animals by 15% when SQ fat was transplanted to the VIS cavity (p<0.01)(). Plasma insulin levels paralleled the glucose levels and were similar among the sham, VIS-VIS, and VIS-SQ groups (about 850 pg/ml), decreased by 26% in the SQ-SQ group and decreased by 33% in the SQ-VIS group when compared to the sham group, although these differences did not quite reach statistical significance. Thus, adding SQ fat to a normal mouse resulted in decreased glucose levels with normal or decreased insulin levels suggesting improved insulin sensitivity. These metabolic improvements were greatest when the SQ fat was transplanted to the VIS cavity and were specific to the addition of SQ fat. Adding VIS fat to the VIS cavity had no effect on body weight gain, body composition, glucose or insulin levels. Plasma leptin levels were not significantly different among the sham, VIS-VIS, VIS-SQ and SQ-SQ groups, but were significantly decreased by 70% in the SQ-VIS in comparison to that of the sham controls (p<0.05). The low leptin levels in the SQ-VIS group correlate with the lower fat mass in this group. Total plasma adiponectin levels were similar between the sham and VIS-VIS groups, significantly decreased by 12% in the VIS-SQ and SQ-SQ groups (p<0.05) and decreased by 25% in the SQ-VIS group (p<0.01) in comparison to the sham group. Higher levels of high molecular weight (HMW) adiponectin to total adiponectin ratio, but not absolute total adiponectin levels, correlate better with improved insulin sensitivity (Pajvani et al., 2004
). However, percent HMW adiponectin to total adiponectin was not significantly different among the sham and all transplantation groups (). Since adiponectin levels in the SQ-VIS group were either decreased or similar to that of the sham group in the SQ-VIS group, it is unlikely that adiponectin can account for the decreased body weight or fat mass in this group.
Figure 3 Basal Plasma Levels of Hormones and Substrates and Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) After Fat Transplantations in Cohort 1. (A) Basal plasma glucose and insulin levels were not significantly different among the VIS transplantation (more ...)
At 10 weeks after transplantation, intraperitoneal glucose tolerance tests were performed in the mice. Most notable was the SQ-VIS group which had the lowest glucose levels in comparison to levels in the sham group, and this reached statistically significance at 120 minutes after the glucose load (p<0.05) (). In the remaining groups, blood glucose levels during the 120-minute period tended to be highest in the VIS-VIS group, similar to the sham group for the VIS-SQ group, and lower in the SQ-SQ group, although these differences did not reach statistical significance. Thus, adding SQ fat to the VIS cavity significantly improved insulin glucose tolerance, whereas adding VIS fat to the VIS cavity, if anything, tended to cause a deterioration of glucose tolerance. Intraperitoneal insulin tolerance tests performed at 11 weeks after transplantation showed no significant difference among the groups, although at 120 min, the SQ-SQ groups tended to have lower glucose levels than the other groups.
Insulin Sensitivity by Hyperinsulinemic-Euglycemic Clamp
To more precisely and directly assess insulin sensitivity, we utilized the hyperinsulinemic-euglycemic clamp coupled with D-[3-3H]-glucose and 14C-deoxyglucose infusions. This allowed assessment of three different parameters of glucose metabolism: 1) whole body insulin sensitivity; 2) glucose uptake into muscle and endogenous and transplanted fat; and 3) effects of insulin on hepatic glucose output. These hyperinsulinemic-euglycemic clamps were performed at 12 weeks after transplantations in cohort 2.
Direct measurement of whole-body insulin sensitivity was quantified during the hyperinsulinemic-euglycemic clamp as the amount of exogenous glucose infusion required to maintain blood glucose levels at initial fasting levels during the hyperinsulinemic infusion and expressed as glucose infusion rate (GIR). GIR was not significantly different between the sham and VIS-VIS groups (). However, the GIR was significantly increased by 2.0-fold when SQ fat was transplanted to the SQ area and had an even greater increase to 2.4-fold when SQ fat was transplanted to the VIS area in comparison to the GIR in the sham group (p<0.05). Hence, increasing SQ fat in the SQ depot and, more strikingly, increasing SQ fat in the VIS depot improved whole-body insulin sensitivity as measured by glucose infusion rate in response to a stable insulin infusion.
Figure 4 Direct Measures of Insulin Sensitivity by Hyperinsulinemic- Euglycemic Clamp After Fat Transplantations in Cohort 2. (A) Whole-body insulin sensitivity (quantified by glucose infusion rate; GIR) was not significantly different among the sham, VIS-VIS, (more ...)
Insulin-stimulated glucose uptake into transplanted fat depots and endogenous fat and muscle was assessed during the final 45 minutes of the hyperinsulinemic-euglycemic clamp using 14C-deoxyglucose. In the sham-operated group, 14C-deoxyglucose uptake was similar in endogenous SQ fat and VIS fat (), i.e., there was no intrinsic difference in glucose uptake between the SQ and VIS fat depots in the control animals of this study. All fat grafts in the three transplantation groups had at least the same level of glucose uptake as the endogenous SQ and VIS fat in the sham group (). In the transplanted fat grafts themselves, 14C-deoxyglucose uptake appeared to be lowest in the VIS-VIS group and tended to be higher in the SQ-SQ and SQ-VIS groups, although these changes were not statistically significant. In fact, the glucose uptake in the fat grafts averaged 1.4- to 5.7-fold higher than that of the fat in the sham group, further indicating good vascularization and function of the transplanted fat grafts (), and thereby confirming the success of the fat transplantation. Most interestingly, 14C-deoxyglucose uptake into endogenous SQ fat of the recipient host mice was significantly increased by about 2.5- to 2.8-fold in the groups with SQ fat transplanted into either the SQ or VIS depots (SQ-SQ and SQ-VIS groups) in comparison to the sham group (p<0.05), whereas endogenous SQ fat in the group with VIS fat transplanted into the VIS cavity had similar levels of glucose uptake as the fat in the sham group (). These results suggest cross-talk between SQ fat grafts and endogenous SQ fat, regardless of whether the SQ fat graft is transplanted to the SQ area or VIS cavity. By contrast, 14C-deoxyglucose uptake into endogenous VIS fat was not different among the sham and three transplantation groups ().
Hepatic glucose production (HGP) was assessed in both the basal state and during the insulin clamp (). Basal HGP was not significantly different between all groups, although it tended to be decreased in the SQ-VIS group in comparison to the sham group. Insulin infusion during the clamps decreased HGP in the sham and VIS-VIS group as expected, but more interestingly, the decrease was greatest in the SQ-SQ and SQ-VIS groups. In the sham and VIS-VIS groups the insulin suppression of HGP was 52±18% and 71±36%, respectively, as compared to their own corresponding basal HGP levels (). Consistent with the improved insulin sensitivity, insulin suppression of HGP was greatest in the groups with SQ fat transplantation with 97±2% suppression in the SQ-SQ group and 100±0% suppression in the SQ-VIS group. 14C-deoxyglucose uptake in muscle was not significantly different among all groups (). Thus, the addition of SQ fat to a normal mouse via transplantation enhanced insulin sensitivity in the liver, as well as on endogenous SQ fat. In all cases, the effect was observed when SQ fat was added to the SQ area but was most pronounced when SQ fat was added to the VIS cavity. By contrast, addition of VIS fat to the VIS cavity did not improve nor worsen insulin sensitivity.
Histology and Markers of Inflammation
At 12 weeks after fat transplantation, the recipient mice were sacrificed, the endogenous fat and exogenous fat grafts, as identified by presence of GFP, were removed for histological examination. In cohort 2, hematoxylin and eosin staining of endogenous visceral/epididymal and subcutaneous/flank fat pads of the recipient mice showed normal appearing signet rings of fat cells, with occasional interspersed macrophages and vascular cells. The transplanted fat allografts had normal histology in comparison to endogenous SQ and VIS fat in the sham-operated group (). The mean area of the adipocytes in the visceral depot appeared to be larger than that of the subcutaneous depot in the sham group by 20%, but this was not statistically significant (i.e. first and third bars in ). Adding visceral fat to the visceral cavity did not significantly change the mean area of the transplanted adipocytes in comparison to that of the endogenous visceral fat in the sham group (i.e. first two bars in ). Adding subcutaneous fat to the subcutaneous area tended to decrease average adipocyte size in comparison to the endogenous SQ fat in the sham group, but this also did not reach statistical significance. Remarkably, transplantation of subcutaneous fat to the visceral cavity (SQ-VIS group) significantly decreased average adipocyte area by 38% when compared to cells in the endogenous subcutaneous fat depot, and more importantly, did not increase in size to that of the surrounding endogenous VIS fat. Vascularization in the transplanted GFP fat grafts was observed in whole-mounts of fat, thereby further confirming the viability of the fat grafts (). Levels of macrophages and inflammation were quantitated by mRNA levels of macrophage cell surface marker F4/80 and the cytokines IL-6 and TNF-α using quantitative real-time qRT-PCR. While all of the transplanted fat had minimally increased levels of mRNA levels for F4/80, IL-6 and TNF-α, this was significant only in the visceral fat transplanted into the visceral depot (). Thus, as expected, the syngenic transplant of fat resulted in very little, if any, rejection or inflammatory reaction.
Figure 5 Histology and Inflammatory Markers in Transplanted and Endogenous Fat in Cohort 2. (A) H&E staining of VIS and SQ fat pads were performed in all groups of cohort 2. Transplanted VIS and SQ fat pads had normal histology in comparison to endogenous (more ...)
To further assess the status of the adipose tissue graft and explore the potential mechanisms by which the adipose graft might affect whole-body metabolism, the expression of several fat-related molecules, specifically PPARγ, FAS, leptin, adiponectin, resistin, and retinol-binding protein (RBP4), was analyzed. Since it was impossible to isolate enough fat cells from the transplanted fat in each mouse for this analysis, the entire transplant or endogenous fat pad was used, and the data were expressed both in terms of absolute mRNA levels and mRNA levels relative to aP2 levels, as a marker for amount of differentiated fat cell present in the tissue. The patterns for absolute gene expression levels and gene expression levels relative to aP2 levels were similar.
This analysis revealed that adding more VIS fat to the VIS area (VIS-VIS group) did not significantly change gene expression levels of PPARγ, FAS, leptin, or adiponectin in the fat graft as compared to levels in the endogenous VIS fat in the sham group, with the exceptions of resistin and RBP4 levels which were 52% and 65% lower, respectively, in the fat grafts than that in the endogenous VIS fat in the sham group (p<0.01) (i.e. comparison of the first and second columns in graphs in ). Also, adding more SQ fat to the SQ area (SQ-SQ group) did not significantly change gene expression levels of the six adipocyte marker genes when compared to endogenous SQ fat in the sham group (i.e. comparison of the third and fourth columns in each graph in ). Thus, adding more fat of the same depot did not change relative gene expression of PPARγ, FAS, leptin, and adiponectin with either the SQ and VIS fat grafts, but did have an effect to lower resistin and RBP4 levels when VIS fat was transplanted to the VIS area but not when SQ fat was transplanted to the SQ area. When SQ fat was transplanted to the VIS area (SQ-VIS group), gene expression levels for PPARγ, FAS, leptin, and RBP4 were also not significantly different from any other group, although the gene expression levels for the fat graft in the SQ-VIS group showed considerable variability. Interestingly, adiponectin and resistin levels in the SQ fat graft that was transplanted to the VIS cavity (SQ-VIS group) were significantly decreased by 2.6- and 2.2-fold, respectively, when compared to the endogenous SQ fat in the sham group (p<0.01 and p<0.05). These low gene expression levels for leptin and adiponectin are consistent with the lower levels of leptin and adiponectin in the plasma of the SQ-VIS group.
Figure 6 Gene Expression in Endogenous and Transplanted fat was measured by real-time qRT-PCR. Analyses of fat-related molecules, such as PPARγ, FAS, leptin, adiponectin, resistin and RBP4, showed that: 1) all transplanted fat grafts (in the VIS-VIS, SQ-SQ, (more ...)
Overall, these analyses of gene expression levels showed that: 1) the transplanted fat grafts in the VIS-VIS, SQ-SQ, and SQ-VIS transplantation groups expressed PPARγ and FAS mRNA levels that were not significantly different from those of endogenous VIS or endogenous SQ fats in the sham group; 2) adding more fat of the same depot (VIS-VIS or SQ-SQ) did not change gene expression levels of PPARγ, FAS, leptin, and adiponectin, except for resistin and RBP4 when VIS fat was added to the VIS cavity; and 3) adding SQ fat to the VIS cavity lowered mRNA levels of leptin, adiponectin and resistin, but not PPARγ, FAS or RBP4, in comparison to levels in the endogenous SQ fat.