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 surgery in the VIS or SQ area, but no fat was transplanted.

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 in the SQ-VIS group in comparison to the sham group. All data are presented as mean ± SEM. (C) Fat mass was similar between the sham and VIS transplantation groups, decreased in the SQ-SQ group, and most reduced in the SQ-VIS group. (D) Percent body fat was significantly lower only in the SQ-VIS group as compared to the sham group. (E) Lean mass and (F) percent lean mass were significantly higher in the SQ-VIS in comparison to the sham group. (G) Total energy expenditure (TEE) (not shown) and TEE divided by lean mass were not significantly different among all sham and transplantation groups during the light and dark cycles. (H) Respiratory Quotient (RQ) was significantly higher in the SQ-VIS group than in the sham group during the light and dark cycles and (I) almost all 30 minute-intervals during the 24-hour measurement, thereby indicating a higher proportion of carbohydrate to fat oxidation in the SQ-VIS group.

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 groups and sham groups, were lower in the SQ-SQ group, and lowest in the SQ-VIS group. Plasma leptin and total adiponectin levels also had a decreasing pattern with levels highest in the sham group and significantly lowest in the SQ-VIS group. (B) Plasma samples were electrophoresed through nonreducing SDS gels, transferred to membranes, and probed with rabbit anti-mouse adiponectin antibody. Percent of high molecular weight (HMW) adiponectin to total (i.e. HMW + medium molecular weight (MMW) + low molecular weight (LMW)) adiponectin was not significantly different among all groups. (C) After a two hour fast for the GTT, glucose was given i.p. Glucose levels at 120 min were significantly lower only in the SQ-VIS group as compared to sham group. After an overnight fast for the ITT, insulin was injected i.p. No significant difference in blood glucose levels was observed among all groups.

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, and SQ-SQ groups, but was significantly higher in the SQ-VIS group. (B) Insulin-stimulated ¹⁴C-deoxyglucose (14C-DG) uptake was similar between the endogenous VIS fat and the endogenous SQ fat in the sham group. 14C-DG uptake in the fat grafts was not significantly different in all three transplantation groups, and had at least the same level of 14C-DG uptake as the endogenous fat. (C) 14C-DG uptake into endogenous SQ fat of the host mice was greater in the SQ-SQ and SQ-VIS groups in comparison to the sham and VIS-VIS groups. (D) 14C-DG uptake into endogenous VIS fat of the sham and all transplantation groups was not significantly different. (E) Hepatic glucose production (HGP) at basal time point was not significantly different among all groups, with a trend for lowest levels in the SQ-VIS group in comparison to the sham group. As expected during the clamp, insulin decreased HGP in the sham and VIS-VIS groups in comparison to their corresponding basal HGP levels, and more importantly, further decreased HGP in the SQ-SQ and SQ-VIS groups. (F) The greatest percent suppression of HGP during the hyperinsulinemic clamp with respect to the basal level was in the SQ-SQ and SQ-VIS groups. (G) Glucose uptake in muscle was not significantly different among all groups.

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 VIS and SQ fat pads in the sham group as shown at 400X magnification. (B) Area of adipocytes was measured on the H&E slides with ImageJ software. Transplanting SQ fat to the visceral cavity (SQ-VIS group) significantly decreased the mean area of the adipocytes in comparison to that of the endogenous SQ fat in the sham group. (C) Whole-mount of transplanted fat graft expressing GFP was viewed under UV light. Arrows indicate sites of vascularization in the fat graft. (D) Presence of macrophages and inflammatory markers was assessed by quantifying mRNA expression levels of macrophage cell surface receptors F4/80 and inflammatory cytokines interleukin-6 and TNF-α by real-time qRT-PCR. Levels in the VIS-VIS fat grafts were significantly higher than that of the endogenous VIS fat in the sham group. There were no significant differences in macrophages, IL-6, or TNF-α among the SQ-SQ and SQ-VIS fat grafts and the endogenous fat pads.

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, and SQ-VIS transplantation groups) express PPARγ and FAS mRNA levels that are not significantly different from that of endogenous VIS or endogenous SQ fats in the sham group; 2) adding more fat of the same depot (VIS-VIS or SQ-SQ) does not change gene expression levels of the first four fat genes analyzed except for resistin and RBP4 in the VIS-VIS group; and 3) adding SQ fat to the VIS cavity lowers mRNA levels of leptin, adiponectin, resistin and RBP4 in comparison to levels in the endogenous VIS and SQ fats in the sham group.