Ectopic brown adipose tissue in muscle provides a mechanism for differences in risk of metabolic syndrome in mice

Abstract

C57BL/6 (B6) mice subjected to a high-fat diet develop metabolic syndrome with obesity, hyperglycemia, and insulin resistance, whereas 129S6/SvEvTac (129) mice are relatively protected from this disorder because of differences in higher basal energy expenditure in 129 mice, leading to lower weight gain. At a molecular level, this difference correlates with a marked higher expression of uncoupling protein 1 (UCP1) and a higher degree of uncoupling in vitro in mitochondria isolated from muscle of 129 versus B6 mice. Detailed histological examination, however, reveals that this UCP1 is in mitochondria of brown adipocytes interspersed between muscle bundles. Indeed, the number of UCP1-positive brown fat cells in intermuscular fat in 129 mice is >700-fold higher than in B6 mice. These brown fat cells are subject to further up-regulation of UCP1 after stimulation with a beta3-adrenergic receptor agonist. Thus, ectopic deposits of brown adipose tissue in intermuscular depots with regulatable expression of UCP1 provide a genetically based mechanism of protection from weight gain and metabolic syndrome between strains of mice.

Fig. 1.

Weight, fat percentage, daily weight gain, and feeding efficiency of B6 and 129 mice. Mice were maintained on either a low-fat diet (14% of calories from fat) or high-fat diet (55% fat) starting by the end of puberty (around the age of 6 weeks). Dual-energy x-ray absortionmetry (DEXA) scanning was performed after ≈6 weeks on the diets. (A) Body weight at the time of DEXA scanning in B6 and 129 mice on low-fat (open bars) and high-fat diets (filled bars). B6 versus 129 mice on low-fat diet, P = 0.043; on high-fat diet, P = 0.021. (B) Fat percentage determined by DEXA scanning of B6 and 129 mice on low- and high-fat diets. B6 versus 129 on low-fat diet, P = 0.021; on high-fat diet, P = 0.029. (C) Average daily weight gain during the 18 weeks on low- and high-fat diets. B6 versus 129 on low-fat diet, P = 0.006; on high-fat diet, P = 0.001. (D) The mice were placed in individual cages, and food was measured every 3rd day. Feeding efficiency was calculated as weight gain per kcal of food intake in mice on both diets. Feeding efficiency was 2.6-fold lower in 129 versus B6 on the low-fat diet (P = 0.021) and 1.3-fold lower in 129 on the high-fat (P = 0.001). This latter result was previously reported as part of an analysis of the genetic determinants of energy expenditure and insulin resistance (6); together with the new data in A–C in this figure and the data in Fig. 2, it demonstrates the full range of metabolic characteristics of diet-induced obesity in this cohort of these two strains of mice. All results are expressed as mean ± SEM with four mice in each group.

Fig. 2.

Activity and metabolic rate of B6 and 129 mice on low- and high-fat diets. Mice were placed in indirect calorimetry chambers and allowed to adapt for 48 h. Activity was measured as beam break counts during 24 h on low-fat (A) and high-fat diets (B) in B6 (filled circles) and 129 mice (open circles). The black horizontal bar denotes the dark period. (C) The bars represent the mean activity level over 24 h ± SEM with four mice in each group. Mean activity level of 129 versus B6 mice was significantly lower (P < 0.001) on both diets.

Fig. 3.

Gene expression in skeletal muscle and expression of fat-specific genes in skeletal muscle and epididymal fat. (A) Gene array analysis of skeletal muscle by using Affychip U72Av.2. The scatter plot depicts the genes and ESTs with differential expression between B6 and 129 of >1.15-fold. The UCP1 gene expression (encircled) exhibited the highest fold difference between the two strains (52-fold higher in 129 versus B6). (B) The UCP1 gene expression of skeletal muscle was confirmed by quantitative RT-PCR showing a 110-fold higher expression of UCP1 in 129 than in B6 (P = 0.0002). Gene expression of UCP1 and of white fat-specific genes (leptin, adiponectin, and aP2) in muscle (C) and epididymal white fat (D) of B6 (filled bars) and 129 mice (open bars) is shown. Only leptin in muscle was significantly higher in B6 mice compared with 129 mice (P = 0.023). Data represent the mean of four chips with cRNA from two or three mice on each chip. The mice used for the study had been maintained on a regular chow (21% of calories from fat) for 6 months.

Fig. 4.

Kinetics of the proton leak in mitochondria isolated from 129 and B6 strain mice. Respiration and mitochondrial inner membrane potential were determined simultaneously in BAT (n = 10 for each strain) (A) and hindlimb muscle (n = 8 for each strain) (B) of B6 (filled squares) and 129 mice (open circles). Data points and error bars represent mean ± SEM for both oxygen use and potential, and they are thus directed along both the x and y axes. Please note that different scales have been used for A and B. The mice used for the study had been maintained on a regular chow.

Fig. 5.

Morphology of adipose tissue from skeletal muscle of 129 and B6 mice. The bones of the posterior leg were removed from 13-week-old mice, and four transverse serial sections of the thigh (A) and three sections of the popliteal region (B) were obtained from B6 (filled bars) and 129 mice (open bars). The percentage of the area of each transverse section occupied by adipose tissue was calculated by using a morphological imaging system (see Experimental Procedures). The adipose tissue mass was significantly higher in 129 compared with B6 mice of the thigh only, as indicated on the figure. (C) A representative staining of UCP1 immunoreactive multilocular adipocytes is shown. (Scale bar, 50 μm.) Quantification of the UCP1-positive adipocytes of the thigh (D) and popliteal region (E) revealed a statistically significantly higher number of UCP1-positive cells as indicated by the P values on the figures. The bars represent the mean ± SEM of five mice of each strain maintained on regular chow at 22°C.

Fig. 6.

Western blot analysis of UCP1. Adipose-free extract (A) or adipose-containing extract (B) of popliteal and quadriceps was used for Western blot analysis, and it was compared with BAT and epididymal fat. Twenty micrograms of protein was loaded in each lane and blotted with UCP1 antibody. The arrows indicate location of bands at 32 kDa, where UCP1 is located.

Fig. 7.

β₃-Adrenergic receptor agonist-induced UCP1 expression. B6 and 129 mice on a regular chow were treated with saline (filled bars) or β₃-adrenergic receptor agonist (open bars) for 7 days. Mice were killed, and UCP1 gene expression was determined by quantitative RT-PCR (see Experimental Procedures) in skeletal muscle (A), epididymal fat (B), s.c. fat (C), and BAT (D). After the 7 days, there was a significantly higher expression of UCP1 in total skeletal muscle of the 129 mice (P = 0.029) compared with B6. Each bar represents the mean ± SEM of eight mice.