Manipulation of Dietary Amino Acids Prevents and Reverses Obesity in Mice Through Multiple Mechanisms That Modulate Energy Homeostasis
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Figures
- Figure 1
EAA, unlike CAA substitution for protein content of rodent diets, prevents and reverses obesity, with the improvement of glucose homeostasis and extension of average life span. A: Body weight of mice fed with the chow, SFA, SFA-EAA, and SFA-CAA diet at room temperature (n = 7–10 mice per group). After maximal body weight was reached (11 months, dashed line), mice fed with the SFA diet were switched to either the SFA-EAA diet (SFA to SFA-EAA) or the SFA-CAA diet (SFA to SFA-CAA) (n = 5 mice per group). B: Body weight of mice at room temperature (n = 9–10 mice per group). C: Body composition at the end of treatment: fat mass (left) and lean mass (right) (n = 6 mice per group). D: Plasma leptin levels at different times at room temperature (n = 5 mice per group). E: Body weight of mice at thermoneutrality (30°C) (n = 10 mice per group). Glucose tolerance test results in mice at room temperature (n = 5 mice per group) (F) or at thermoneutrality (G) (n = 7 mice per group). Mice were fed with different diets for 6 weeks. Plasma levels of adiponectin (H) and IGF-1 (I) in mice fed with the SFA-CAA or SFA-EAA diet for 6 weeks at room temperature (n = 5 mice per group). J: Kaplan-Meier survival curves for chow, SFA-, SFA-EAA–, and SFA-CAA–fed mice (n = 35 mice per group). All data (except J) are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. SFA diet; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. SFA-CAA diet; §P < 0.05, §§P < 0.01, and §§§P < 0.001 vs. chow diet.
- Figure 2
Food intake and energy absorption in mice on different dietary regimens. A: Food intake of mice fed with chow, SFA, SFA-EAA, and SFA-CAA diets for different time intervals (n = 5–7 mice per group). B: Gut transit time. C: Hematoxylin and eosin staining of jejunum villi. Scale bar, 90 μm. D: Energy excretion: the mean amount of feces excreted per mouse in 24 h (mg/24 h) (left) and food excreted (dry mass of fecal pellets, collected over 72 h and expressed as the percentage of food intake) (right). E: Mean energy content in feces (kcal/g). F: Daily energy excretion (kcal/mouse) calculated using the previous values (n = 3–5 mice per group). B–F: Mice were fed with the SFA, SFA-CAA, and SFA-EAA diets for 6 weeks. All data are presented as mean ± SEM. **P < 0.01 and ***P < 0.001 vs. SFA diet; ###P < 0.001 vs. SFA-CAA; §§§P < 0.001 vs. chow diet.
- Figure 3
Energy metabolism in mice on different dietary regimens. A: Metabolic efficiency was calculated as the body weight (BW) gain-to-the energy intake ratio (i.e., total food consumed during 5 days or 2 or 6 weeks). EE (B) and RER (C) during one 24-h cycle. D: Whole-body temperature measured with a digital rectal thermometer. Measurements were performed in two separate experiments after 2 or 6 weeks (n = 5–7 mice per group). All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. SFA-CAA diet.
- Figure 4
SFA-EAA diet changes gut microbiota composition. A: Chao index, Shannon entropy index, and α-diversity calculated on the number of observed operational taxonomic units (OTUs). B: Principal coordinates analysis (PCoA) of bacterial β-diversity based on the Bray-Curtis dissimilarity index. Each symbol represents a single sample of feces after 6 weeks of treatment. C: Box-and-whisker plot of intercommunity β-diversity determined by the Bray-Curtis dissimilarity index. D: Phylum/order level relative abundance expressed as geometric mean. E: Allobaculum, Sutterella, and Lactococcus relative abundance. F: Random forest analysis. For the box-and-whisker plots, the boxes show median and first and third quartiles. The whiskers extend from the quartiles to the last data point within 1.5× interquartile range, with outliers beyond represented as dots (n = 10 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 as shown.
- Figure 5
The SFA-EAA diet promotes uncoupled respiration in iWAT without browning stimulation. A: Uncoupled (i.e., with oligomycin) and maximal (i.e., with FCCP) OCRs in iWAT mitochondria; respiration was normalized to mitochondrial protein amount (n = 5 mice per group). B: Western blot analysis of UCP1 and HSP60 protein levels in adipose tissues (n = 4 mice per group). One experiment representative of three reproducible ones is shown. iBAT of mice fed with the chow diet at room temperature was used as the control. C: Thermogenic N-acyl amino acids in plasma (left) and iWAT (right) (n = 4–5 mice per group). D: Relative mRNA levels of the Pm20d1 gene in different tissues (n = 5 mice per group). A–D: Mice were fed with the SFA-CAA and SFA-EAA diet for 6 weeks at thermoneutrality. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. SFA-CAA diet.
- Figure 6
SFA-EAA diet increases the thermogenic function of iBAT. A: iBAT temperature was measured with a thermographic FLIR camera in mice fed with the SFA-CAA and SFA-EAA diet for 6 weeks at room temperature (n = 5 mice per group). B: Uncoupled (i.e., ΔGDP, UCP1-dependent respiration calculated as the amount of ADP-independent respiration that was inhibited by GDP) and maximal (FCCP) OCRs in iBAT mitochondria; respiration was normalized to mitochondrial protein amount (n = 5 mice per group). C: Western blot analysis of UCP1 and GAPDH protein levels in iBAT. One experiment representative of three reproducible ones is shown (n = 3–5 mice per group). Mice in B and C were fed with the SFA-CAA and SFA-EAA diet for 1, 5, or 20 days at room temperature. D: Uncoupled (ΔGDP) and maximal (FCCP) OCR in iBAT mitochondria (n = 6 mice per group). E: Western blot analysis of UCP1, cytochrome c oxidase subunit IV (COX IV), cytochrome c (Cyt c), and GAPDH protein levels in iBAT. One experiment representative of three reproducible ones is shown (n = 4 mice per group). F: Electron microscopy analysis of iBAT. Scale bar, 0.5 μm (n = 2 mice per group). Mice in D–F were fed with the SFA-CAA and SFA-EAA diet for 6 weeks at room temperature. All data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 vs. SFA-CAA diet.
- Figure 7
The SFA-EAA diet activates brown adipocytes. A: NE turnover (NETO) in iBAT of mice fed with the SFA-CAA and SFA-EAA diet for 6 weeks at room temperature. NETO was assessed by NE synthesis inhibition with α-methyl-p-tyrosine (n = 5 mice per group). B: Relative mRNA levels of noradrenergic innervation markers in iBAT of mice fed with the SFA-CAA and SFA-EAA diet for 6 weeks. C: Scheme of the in vitro experiment in primary brown adipocytes isolated from mice fed with the chow diet. After 16-h incubation in amino acid-free medium, brown adipocytes were pretreated with rapamycin (Rapa, 1.0 nmol/L) or vehicle (Veh; DMSO) for 1 h. Then, the cells were supplemented with Veh (PBS) or CAA mixture (CAAm) or EAA mixture (EAAm), specifically reproducing the iBAT aminograms resulting from consumption of either the SFA-CAA or SFA-EAA diets, respectively, as reported in Supplementary Table 4 (n = 3 experiments performed in triplicate). D: Ucp1 mRNA levels in differentiated brown adipocytes, treated as in C. Western blot analysis of UCP1 and GAPDH (E), and (Ser 235/236) phosphorylated (p) S6 and S6 (G) protein levels in differentiated brown adipocytes, treated as in C. One immunoblot experiment representative of three reproducible ones is shown. F: OCRs of differentiated brown adipocytes, treated as in C. OCR was measured with the Seahorse XF24 Extracellular Flux Analyzer (n = 3 readings in quadruplicate per group). All data are presented as mean ± SEM. **P < 0.01 and ***P < 0.001 vs. Veh; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. CAAm; §P < 0.05 and §§§P < 0.001 vs. EAAm + Rapa. TH, tyrosine hydroxylase; β3AR, β3-adrenergic receptor.
- Figure 8
mTORC1 signaling contributes to iBAT thermogenesis induced by the SFA-EAA diet. A: Western blot analysis of (Ser 235/236) phosphorylated (p) S6 and S6 protein levels in iBAT of mice fed with the SFA-CAA and SFA-EAA diet for different time intervals at room temperature. One immunoblot experiment representative of three reproducible ones is shown (n = 3–5 mice per group). B: Western blot analysis of the mTORC1 pathway in iBAT of mice fed with the SFA-CAA and SFA-EAA diet, with vehicle (Veh) or rapamycin (Rapa) (n = 4 mice per group). C: UCP1-dependent (ΔGDP) and maximal (FCCP) OCRs in iBAT mitochondria. OCR was normalized to mitochondrial protein amount (n = 5 mice per group). D: Rectal and thermographic measurement of iBAT temperature (n = 5 mice per group). E: Western blot analysis of UCP1 protein levels in iBAT. Mice in B–E were fed with the SFA-CAA or SFA-EAA diet for 6 weeks at room temperature, with or without rapamycin (i.p. 2.5 mg/kg body wt) delivered in 200 μL, 5 days per week for 6 weeks, starting with diets (n = 5–6 mice per group). All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. SFA-CAA diet; #P < 0.05 vs. SFA-EAA diet. 4E-BP1, eukaryotic initiation factor 4E-binding protein 1.
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