Leucine deprivation decreases fat mass by stimulation of lipolysis in white adipose tissue and upregulation of uncoupling protein 1 (UCP1) in brown adipose tissue

Abstract

Objective: White adipose tissue (WAT) and brown adipose tissue (BAT) play distinct roles in adaptation to changes in nutrient availability, with WAT serving as an energy store and BAT regulating thermogenesis. We previously showed that mice maintained on a leucine-deficient diet unexpectedly experienced a dramatic reduction in abdominal fat mass. The cellular mechanisms responsible for this loss, however, are unclear. The goal of current study is to investigate possible mechanisms.

Research design and methods: Male C57BL/6J mice were fed either control, leucine-deficient, or pair-fed diets for 7 days. Changes in metabolic parameters and expression of genes and proteins related to lipid metabolism were analyzed in WAT and BAT.

Results: We found that leucine deprivation for 7 days increases oxygen consumption, suggesting increased energy expenditure. We also observed increases in lipolysis and expression of beta-oxidation genes and decreases in expression of lipogenic genes and activity of fatty acid synthase in WAT, consistent with increased use and decreased synthesis of fatty acids, respectively. Furthermore, we observed that leucine deprivation increases expression of uncoupling protein (UCP)-1 in BAT, suggesting increased thermogenesis.

Conclusions: We show for the first time that elimination of dietary leucine produces significant metabolic changes in WAT and BAT. The effect of leucine deprivation on UCP1 expression is a novel and unexpected observation and suggests that the observed increase in energy expenditure may reflect an increase in thermogenesis in BAT. Further investigation will be required to determine the relative contribution of UCP1 upregulation and thermogenesis in BAT to leucine deprivation-stimulated fat loss.

FIG. 1.

Body weight and fat mass decreases in leucine-deprived mice. Mice were fed a control, leucine-deficient, or pair-fed diet for 7 days. Body weight and food intake were monitored daily. Data are means ± SE of at least two independent experiments with mice of each diet for each experiment (control diet, n = 6; (−) leu diet, n = 6; pair-fed diet, n = 6). Statistical significance was determined by one-way ANOVA followed by the Student-Newman-Keuls test for the effect of either (−) leu or pair-fed diet versus control diet (*P < 0.01) or (−) leu diet versus pair-fed diet (#P < 0.01). A: Food intake change. (In every case, pair-fed mice consumed all of this food every day. For this reason, there are no error bars for food intake in this group.) B: Body weight reduction. C: Adipose tissue mass in proportion to body weight. D and E: Body composition measured with nuclear magnetic resonance.

FIG. 2.

Leucine deprivation increases energy expenditure. The energy expenditure was measured by indirect calorimetry in mice fed a control, leucine-deficient, or pair-fed diet for 7 days. A: 24-h oxygen consumption. B: RER. An RER of 0.70 indicates that fat is the predominant fuel source; an RER of 0.85 suggests a mix of fat and carbohydrates, and a value of ≥1.00 is indicative of carbohydrates being the predominant fuel source. C: Rectal temperature. D: Physical activity. Data are means ± SE of at least two independent experiments with mice of each diet for each experiment (control diet, n = 6; (−) leu diet, n = 6; pair-fed diet, n = 6) over 24–48 h after 6-h acclimation to the metabolic chamber. Statistical significance was determined by one-way ANOVA followed by the Student-Newman-Keuls test for the effect of either (−) leu or pair-fed diet versus control diet (*P < 0.01) or (−) leu diet versus pair-fed diet (#P < 0.01).

FIG. 3.

Cell volume diminishes in WAT of leucine-deprived mice. WAT histology for mice fed a control, leucine-deficient, or pair-fed diet for 7 days is shown. A: WAT sections from mice in each group were stained with hematoxylin and eosin (×20 magnification). Images shown are representative of several animals for each group. B: Analysis of WAT cell volume. C: DNA content of total abdominal WAT. Data are means ± SE of at least two independent experiments with mice of each diet for each experiment (control diet, n = 6; (−) leu diet, n = 6; pair-fed diet, n = 6). Statistical significance was determined by one-way ANOVA followed by the Student-Newman-Keuls test for the effect of either (−) leu or pair-fed diet versus control diet (*P < 0.01) or (−) leu diet versus pair-fed diet (#P < 0.01).

FIG. 4.

Triglyceride lipolysis and fatty acid β-oxidation genes increase in WAT of leucine-deprived mice. Expression of triglyceride lipolysis in WAT of mice fed a control, leucine-deficient, or pair-fed diet for 7 days is shown. Data are means ± SE of at least two independent real-time PCR experiments (D and E) or Western blot (A and F) with mice of each diet for each experiment (control diet, n = 6; (−) leu diet, n = 6; pair-fed diet, n = 6). Statistical significance was determined by one-way ANOVA followed by the Student-Newman-Keuls test for the effect of either (−) leu or pair-fed diet versus control diet (*P < 0.01) or (−) leu diet versus pair-fed diet (#P < 0.01). A: p-HSL, HSL, and p-PKA substrate proteins. B: Glycerol release assay. C: cAMP content. D: Adrb3 mRNA. E: Pparα, Cpt-1, and Aco1 mRNA. F: PPARα protein.

FIG. 5.

Lipogenic genes are repressed in WAT of leucine-deprived mice. Expression of lipogenic genes in WAT of mice fed a control, leucine-deficient, or pair-fed diet for 7 days is shown. Data are means ± SE of at least two independent real-time PCR experiments (A) or Western blot (B and D) with mice of each diet for each experiment (control diet, n = 6; (−) leu diet, n = 6; pair-fed diet, n = 6). Statistical significance was determined by one-way ANOVA followed by the SNK test for the effect of either (−) leu or pair-fed diet versus control diet (*P < 0.01) or (−) leu diet versus pair-fed diet (#P < 0.01). A: Fas, Scd1, AccI, and Me mRNAs. B: FAS protein (top, Western blot; bottom, FAS protein relative to actin and normalized to control diet group). C: FAS enzyme activity. D: SREBP-1c protein (top, Western blot; bottom, SREBP-1c protein relative to actin and normalized to control diet group).

FIG. 6.

Expression of β-oxidation genes and UCP1 mRNA and protein increases in BAT of leucine-deprived mice. Expression of β-oxidation genes and UCP1 mRNA and protein in BAT of mice fed a control, leucine-deficient, or pair-fed diet for 7 days. Data are means ± SE of at least two independent real-time PCR experiments with mice of each diet for each experiment (control diet, n = 4; (−) leu diet, n = 4; pair-fed diet, n = 4). Statistical significance was determined by one-way ANOVA followed by the Student-Newman-Keuls test for the effect of either (−) leu or pair-fed diet versus control diet (*P < 0.01) or (−) leu diet versus pair-fed diet (#P < 0.01). A: Pparα, Cpt1α, Aco, Fatp1, and Lpl mRNA. B: Ucp1 mRNA. C: UCP1 protein. D: Oxygen consumption in isolated BAT. E: Pgc1α, C/ebpα, and Pparγ mRNA. F: Adrb1, Adrb3, and Dio2 mRNA.