Figure 1.
Leucine supplementation protects from HF-induced body weight gain and adiposity by increasing energy expenditure and fatty acid oxidation.
(A) Body weight curve and (B) mean daily food intake of mice exposed to HFD or chow and supplemented or not with leucine in drinking water (n = 10–11 per group). (C) Fat mass and (D) lean mass at week 0 (baseline), as well as at weeks 7 and 15 of the study (n = 10–11 per group). (E) Feed efficiency in chow-fed and HFD-fed mice supplemented or not with leucine in drinking water (n = 10–11 per group). Energy expenditure (F), respiratory quotient (G) and locomotor activity (H) in HFD-fed mice supplemented or not with leucine in drinking water (n = 6–8 per group). Arrows in (A) indicates when in vivo experiments were carried out. EE: energy expenditure analysis; MRI: magnetic resonance imaging whole-body composition analysis; RQ: respiratory quotient. *p<0.05 vs. HFD-water group; #p<0.05 vs. chow-fed groups, §p<0.05 vs. dark phase.
Figure 2.
Plasma leucine levels and molecular changes induced by leucine supplementation in HFD-fed mice.
(A) Plasma amino acids and (B) acylcarnitines deriving from leucine metabolism measured during the fed state in HFD-fed mice supplemented or not with leucine in drinking water (n = 9 per group). (C and E) mRNA expression levels of several markers of mitochondrial activity, fatty-acid metabolism and adrenergic β-receptor subtypes in the BAT (C) and WAT (E) of HFD-fed mice supplemented or not with leucine in drinking water (n = 5 per group). (D) Protein expression of UCP-3 in the BAT of HFD-fed mice supplemented or not with leucine in drinking water (n = 5 per group), β-actin loading control. C5: isovalerylcarnitine; C5-OH (C3-DC-M): hydroxyl-isovalerylcarnitine; C5-MDC: methylglutarylcarnitine; FC: fold change. *p<0.05 vs. HFD-water group.
Table 1.
Plasma lipid levels in HFD-water and HFD-Leu mice (N = 6). Data reported are expressed as mean±SEM.
Figure 3.
Leucine supplementation in HF-fed mice improves insulin sensitivity.
(A) Glucose tolerance test and (B) insulin tolerance test carried out in HFD- or chow-fed mice supplemented or not with leucine in drinking water (n = 10-11 per group). (C) Fasting plasma glucose, (D) fasting plasma insulin and (E) HOMA index measured in mice on HFD supplemented or not with leucine in drinking water (n = 10-11 per group). *p<0.05 vs. HFD-water group; #p<0.05 vs. chow-fed groups.
Figure 4.
Leucine supplementation in HF-fed mice increases intestinal gluconeogenesis and glucose-stimulated insulin release.
(A) G6Pase activity in the jejunum of HFD-fed mice supplemented or not with leucine in drinking water (n = 6 per group). (B) Representative images of Langerhans islets (in brown: insulin staining), (C) islet area and (D) islet insulin content from HFD-fed mice supplemented or not with leucine in drinking water (n = 5 per group). (E) Glucose-stimulated insulin secretion from Langerhans islets of HFD-fed mice supplemented or not with leucine (n = 3 mice per group, two independent experiments). Data are relative to 11 mM secretion in HFD-water mice that was considered 100%. *p<0.05 vs. HFD-water group; #p<0.05 vs. 3 mM condition in each group; §p<0.05 vs. all other groups. Scale bar in (B): 100 µm.
Figure 5.
Reduced adiposity is sufficient to improve insulin sensitivity.
(A) Fat mass, expressed as % of body weight, in HFD-fed mice supplemented or not with leucine and in HFD-pairfat mice (n = 10–11 per group). (B) Energy expenditure in HFD-fed water and pairfat mice (n = 6 per group). (C) Glucose tolerance and (D) insulin tolerance tests carried out in HFD-fed mice supplemented or not with leucine and in pairfat mice. Data are expressed as % of the HFD-water group (n = 10–11 per group). (E) G6Pase activity measured in the jejunum of HFD-fed water and pairfat mice (n = 6 per group). (F) Glucose-stimulated insulin secretion from Langerhans islets of HFD-fed water and pairfat mice (n = 3 mice per group, two independent experiments). Data are relative to 11 mM secretion in HFD-water mice that was considered 100%. *p<0.05 vs. HFD-water group; #p<0.05 vs. 3 mM condition in each group.
Figure 6.
Leucine supplementation in already obese mice does not affect body weight, adiposity or energy expenditure.
(A) Body weight curve, (B) mean daily food intake, (C) fat mass and (D) lean mass composition and (E) 24-h energy expenditure in obese mice supplemented or not with leucine in drinking water while maintained HFD (n = 6–7 mice per group). Arrows in (A) indicates when in vivo experiments were carried out. EE: energy expenditure analysis; MRI: magnetic resonance imaging whole-body composition analysis.
Figure 7.
Leucine supplementation in already obese animals does not affect glucose homeostasis.
(A) Glucose tolerance and (B) insulin tolerance tests carried out in obese mice respectively after 13 weeks and 14 weeks of supplementation or not with leucine in drinking water (n = 7per group).