Figure 3. MMA mice display cold intolerance associated with abnormal brown fat pathology and function.

(A) Sets of 4- to 5-month-old mutant mice, Mmut^–/– Tg^INS-Alb-Mmut, depicted as (–/–) (+), and heterozygous littermates (+/–) (+) exposed to high-protein diet for 2 months were euthanized and their interscapular brown adipose tissue (BAT) sectioned and H&E-stained. Mutant mice display markedly abnormal BAT with decreased cellular content (fewer nuclei) and large lipid droplets as opposed to controls (scale bars: 100 μm). Ultrastructural studies demonstrated multinodular lipid droplets and numerous mitochondria with normal cristae in BAT from heterozygous mice, as opposed to large lipid lobules and sparse mitochondria with abnormal density and cristae in the mutant animals (scale bars: 1 μm). (B) Effects of acute β₃-adrenergic stimulation on energy expenditure. Mmut^–/– Tg^INS-Alb-Mmut showed a 50% lower increase in their metabolic rate in response to a single dose of β-agonist, CL-316,243, compared with their control littermates (P = 0.0016, 1-way ANOVA with Bonferroni correction). (C) Quantitative RT-PCR results showing fold-change mRNA expression for PpargC-1α, Ucp1, and Dio2 at room temperature and after cold exposure (4°C for 3 hours). PpargC-1α showed increased transcription after cold exposure, albeit to a lesser degree than in WT animals in both mutant and heterozygous mice, while Ucp1 and Dio2 had lower expression at baseline and showed no to little change with cold stress. (D) Sagittal and transaxial micro-PET images of mice, reared on high-protein chow for 2 months, after injection with ¹⁸F-fluoro-d-glucose (¹⁸F-FDG). White arrowheads point to the interscapular BAT. Mutant mice had significantly decreased uptake in the BAT, while they showed high uptake in the heart tissue (yellow arrows, bottom panels), compared with heterozygotes. %ID/g, injected dose%/gram. (E) Biodistribution of radioactive label in various tissues. *P < 0.01, **P < 0.001, ***P < 0.0001, and ****P < 0.00001.