Figure 5. Loss of function of Slc6a2 in SAMs rescues thermogenic capacities of ob/ob mice.
(a) Schematic representation of bone marrow transplant from either Slc6a2-/- or control B6 (CD45.1) mice into genetically obese ob/ob mice (ob/obSlc6a2-/- and ob/obCtrl chimeras, respectively). (b) Rectal temperature of ob/obCtrl (black) and ob/obSlc6a2-/- (green) chimeras was measured at RT and after 2 hours of cold challenge (4° C). Each data point represents one mouse. n = 4 mice for ob/obSlc6a2-/-, n = 6 mice for ob/obCtrl, *P = 0.025, ****P < 0.0001. (c) Serum NE levels of the ob/obCtrl (black) and ob/obSlc6a2-/- (green) chimeras were measured at RT and after 2 hours of cold exposure (4°C). Each data point represents one mouse. n = 4 mice per group for ob/obSlc6a2-/-, n = 5 mice per group for ob/obCtrl, *P = 0.022, **P = 0.0072. (d) Optical micrographs of BAT removed from ob/ob chimeras following 2 hours of cold challenge (4° C) and stained with H&E. Left panel represents BAT from ob/obCtrl and right panel shows BAT from the ob/obSlc6a2-/- chimeras. Images are representative of fat organs collected from 4 mice (ob/obCtrl) or 6 mice (ob/obSlc6a2-/-). (e) Expression of mRNA for Ucp1 determined by qRT-PCR and relative to Gapdh in BAT (left panel) and sWAT (right panel) dissected after 2 hours of cold (4° C) challenge. Ob/obCtrl chimeras are represented in black and ob/obSlc6a2-/- chimeras are shown in green. Each data point represents one mouse. n = 4 mice for ob/obSlc6a2-/-, n = 5 mice for ob/obCtrl, *P = 0.0269; **P = 0.0015. (f) Optical micrographs of BAT from ob/obCtrl (left) and ob/obSlc6a-/- (right) chimeras following 2 hours of cold (4° C) challenge and stained with anti-UCP1 antibody. Images are representative of fat organs collected from 4 mice (ob/obCtrl) or 6 mice (ob/obSlc6a2-/-). (g) Optical micrographs of sWAT from ob/obCtrl (left) and ob/ob SAMSlc6a2-/- (right chimeras removed from ob/ob chimeras following 2 hours of cold (4° C) challenge and stained with anti-UCP1 antibody. Images are representative of fat organs collected from 4 mice (ob/obCtrl) or 6 mice (ob/obSlc6a2-/-). (h) Average adipocyte diameter quantified from optical micrographs of sWAT and BAT from ob/ob chimeras following 2 hours of cold (4° C) challenge. Measurements are representative of 4 independent micrographs (4 ob/obSlc6a2-/- mice) or 6 micrographs (6 ob/obCtrl mice). 18-34 measurements were obtained per micrograph. n = 169 for ob/obCtrl sWAT, n = 120 for ob/obSlc6a2-/- sWAT, n = 180 for ob/obCtrl BAT, n = 120 for ob/obSlc6a2-/- BAT, ****P < 0.0001. (i) Body weight change (upper panel) and daily food intake (lower panel) of ob/obCtrl (n = 4 mice) and ob/obSlc6a2-/- (n = 6 mice) chimeras monitored for 7 weeks following 2 weeks of food intake normalization (3 gr/day – grey shade) that started 9 weeks after bone marrow transplant. Yellow triangle indicates when irradiation was performed. *P < 0.05. (j) Blood plasma non-esterified (free) fatty acid concentration in control ob/obCtrl and ob/obSlc6a2-/- chimeras measured 8 weeks after bone marrow transplant and during a 3 gr/day regimen. n = 5 mice per group, **P = 0.0022. Data in panels b, c, e, h, i were analyzed by two-tailed unpaired Student’s t-test and in panel i by multiple t-tests – Student’s t-test per row with correction for multiple comparisons using the Holm-Sidak method. Data are shown as average ± SEM. Scale bars in d, f and g, 100 μm.