UCP1 is an essential mediator of the effects of methionine restriction on energy balance but not insulin sensitivity

Abstract

Dietary methionine restriction (MR) by 80% increases energy expenditure (EE), reduces adiposity, and improves insulin sensitivity. We propose that the MR-induced increase in EE limits fat deposition by increasing sympathetic nervous system-dependent remodeling of white adipose tissue and increasing uncoupling protein 1 (UCP1) expression in both white and brown adipose tissue. In independent assessments of the role of UCP1 as a mediator of MR's effects on EE and insulin sensitivity, EE did not differ between wild-type (WT) and Ucp1(-/-) mice on the control diet, but MR increased EE by 31% and reduced adiposity by 25% in WT mice. In contrast, MR failed to increase EE or reduce adiposity in Ucp1(-/-) mice. However, MR was able to increase overall insulin sensitivity by 2.2-fold in both genotypes. Housing temperatures used to minimize (28°C) or increase (23°C) sympathetic nervous system activity revealed temperature-independent effects of the diet on EE. Metabolomics analysis showed that genotypic and dietary effects on white adipose tissue remodeling resulted in profound increases in fatty acid metabolism within this tissue. These findings establish that UCP1 is required for the MR-induced increase in EE but not insulin sensitivity and suggest that diet-induced improvements in insulin sensitivity are not strictly derived from dietary effects on energy balance.

Keywords: FGF21; adipose tissue; amino acid; housing temperature; obesity.

Figure 1.

Assessment of components contributing to variation in EE. The relative contributions of model components in accounting for variation in total EE is shown as the t ratio of each variable’s impact on EE. EE was measured in mice (n = 7–8) by indirect calorimetry after consuming control (CON) or MR diets for 8 wk at 23°C or 28°C.

Figure 2.

Effect of diet and housing temperature on UCP1 expression. Ucp1 gene and protein expression in BAT (A and B, respectively) and IWAT (C and D, respectively) from WT mice fed the control (CON) or MR diets housed at either 23°C or 28°C for 11 weeks. mRNA expression was normalized to cyclophilin. Relative band densities of UCP1 protein expression were normalized to PDC-E2 (BAT) or β-actin (IWAT). Means ± sem are presented. Different letters denote statistically significant differences.

Figure 3.

Effect of genotype, diet, and housing temperature on adipose tissue morphology. A) Representative H&E stains of IWAT from WT and Ucp1^−/− mice fed the control (CON) or MR diets housed at either 23°C or 28°C for 11 weeks. Scale bars, 400 µm. B) Adipocyte sizes (area per adipocyte, µm²) in control (CON) or MR-fed WT and Ucp1^−/− mice graphed as percent of total adipocytes. X axis indicates bin center with bin sizes of 1000. Means ± sem are presented for each variable and are based on n = 4 per group. Different letters denote statistically significant differences within each adipocyte size bin.

Figure 4.

Effect of genotype, diet, and housing temperature on browning of WAT. A) Representative images of IWAT from WT and Ucp1^−/− mice fed the control (CON) or MR diets housed at either 23°C or 28°C for 11 weeks. Scale bars, 400 µm. Cell membranes were stained with wheat germ agglutinin (green), lipid droplet membranes were stained with perilipin (red), and nuclei were stained with DAPI (blue). B) Relative number of multilocular, brownlike adipocytes graphed as percentage of total adipocytes. Means ± sem are presented for each variable and are based on n = 4 per group. Different letters denote statistically significant differences within each housing temperature.

Figure 5.

Effect of genotype, diet, and housing temperature on FGF21. Hepatic Fgf21 mRNA expression (A) and serum concentrations (B) from WT and Ucp1^−/− mice fed the control (CON) or MR diets housed at either 23°C or 28°C for 11 weeks. mRNA expression was normalized to cyclophilin. Serum samples were collected after a 4-hour food withdrawal. Means ± sem are presented for each variable and based on n = 7–8 per group. Different letters denote statistically significant differences.

Figure 6.

Group contrasts are represented as mean ratios, with red- and green-shaded cells indicating significant (P ≤ 0.05) increases (red) or decreases (green) in the mean ratios. Cells shaded with light red and light green indicate that the mean ratios were trending (e.g., 0.05 < P < 0.10) higher (light red) or lower (light green) for that comparison. For ANOVA of main effects, blue-shaded cells indicate P ≤ 0.05, while light blue-shaded cells indicate 0.05 < P < 0.10.

Figure 7.

Hyperinsulinemic-euglycemic clamps in WT and Ucp1^−/− mice housed at 23°C after 9 weeks of dietary MR. The clamp procedures were conducted as described in the Materials and Methods. A) GIR required to maintain euglycemia during the insulin clamps. B) The ability of insulin to suppress hepatic glucose production (% suppression of endo R_a) during the clamp procedure. Different letters denote statistically significant differences. C) R_g, an indication of tissue-specific insulin-dependent glucose uptake. Means ± sem are presented for each variable and are based on n = 6–9 per group. *Means differ from mice of the same genotype fed the control (CON) diet at P < 0.05.