Diabetes Page 2 of 75

Manipulation of dietary amino acids prevents and reverses in mice through multiple mechanisms that modulate

energy homeostasis

Chiara Ruocco,1 Maurizio Ragni,1 Fabio Rossi,1 Pierluigi Carullo,2 Veronica Ghini,3 Fabiana

Piscitelli,4 Adele Cutignano,4 Emiliano Manzo,4 Rafael Maciel Ioris,5,6 Franck Bontems,5,6 Laura

Tedesco,1 Carolina Greco,2 Annachiara Pino,7 Ilenia Severi,8 Dianxin Liu,9 Ryan P. Ceddia,9

Luisa Ponzoni,1,10 Leonardo Tenori,11,12 Lisa Rizzetto,13 Matthias Scholz,13 Kieran Tuohy,13

Francesco Bifari,1,14 Vincenzo Di Marzo,4 Claudio Luchinat,3,15 Michele O. Carruba,1 Saverio

Cinti,8 Ilaria Decimo,7 Gianluigi Condorelli,2 Roberto Coppari,5,6 Sheila Collins,9 Alessandra

Valerio,16 and Enzo Nisoli1

1Center for Study and Research on Obesity, Department of Biomedical Technology and

Translational Medicine, University of Milan, Milan, Italy; 2Humanitas University, Rozzano,

Italy; 3Interuniversity Consortium for Magnetic Resonance, Sesto Fiorentino, Italy; 4Institute of

Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy; 5Department of

Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland; 6Diabetes Center

of the Faculty of Medicine, University of Geneva, Geneva, Switzerland; 7Department of

Diagnostics and Public Health, University of Verona, Verona, Italy; 8Department of

Experimental and Clinical Medicine, University of Ancona (Politecnica delle Marche), Ancona,

Italy; 9Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University

Medical Center, Nashville, Tennessee, USA; 10Umberto Veronesi Foundation, Milan, Italy;

11FiorGen Foundation, Sesto Fiorentino, Italy; 12Center of Magnetic Resonance, University of

Diabetes Publish Ahead of Print, published online August 19, 2020 Page 3 of 75 Diabetes

Florence, Sesto Fiorentino, Italy; 13Department of Food Quality and Nutrition, Research and

Innovation Center, Fondazione Edmund Mach, San Michele all'Adige, Italy; 14Laboratory of Cell

Metabolism and Regenerative Medicine, Department of Medical Biotechnology and

Translational Medicine, University of Milan, Milan, Italy; 15Department of Experimental and

Clinical Medicine, University of Florence, Florence, Italy; 16Department of Molecular and

Translational Medicine, Brescia University, Brescia, Italy

Corresponding author:

Enzo Nisoli

Department of Biomedical Technology and Translational Medicine

University of Milan, Milan – 20129 (Italy)

Telephone number: +30 02 50316956; Email: [email protected]

C.R. and M.R. contributed equally to this work.

Short title: Dietary amino acids and energy metabolism

The manuscript contains: 6,689 words, 8 figures, 13 supplementary figures, and 10

supplementary tables.

Tweet:

A designer protein-deprived diet enriched in essential amino acids promotes brown fat

thermogenesis, stimulates uncoupling protein 1-independent respiration in white fat, changes the

gut microbiota composition and prevents and reverses obesity, extending healthspan.

Figure 3A best summarizes our article

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Reduced activation of energy metabolism increases adiposity in humans and other mammals. Thus, exploring dietary and molecular mechanisms able to improve energy metabolism is of paramount medical importance, as such mechanisms can be leveraged as a therapy for obesity and related disorders. Here, we show that a designer protein-deprived diet enriched in free essential amino acids can i) promote the brown fat thermogenic program and fatty acid oxidation, ii) stimulate uncoupling protein 1 (UCP1)- independent respiration in subcutaneous white fat, iii) change the gut microbiota composition, and iv) prevent and reverse obesity and dysregulated glucose homeostasis in multiple mouse models, prolonging the healthy lifespan. These effects are independent of unbalanced amino acid ratio, energy consumption, and intestinal calorie absorption. A brown fat-specific activation of the mechanistic target of rapamycin complex 1 seems involved in the diet-induced beneficial effects, as also strengthened by in vitro experiments.

Hence, our results suggest that brown and white fat may be targets of specific amino acids to control UCP1-dependent and -independent thermogenesis, thereby contributing to the improvement of metabolic health.

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Although debatable, chronic consumption of diets with a high ratio of saturated to unsaturated

fatty acids (SFA diets) increases body adiposity, impairs glucose homeostasis, raises the

cardiovascular risk, and reduces the healthy disease-free lifespan in animals and humans (1,2).

These diets negatively affect the proper secretion and action of key hormones—insulin, leptin, and

adiponectin—underlying metabolic homeostasis and energy balance (3). The increased

consumption of high-calorie, low-fiber diets has contributed significantly to the staggering rise in

overweight, obesity, and prevalence in the past few decades (4). Likewise, high-

protein diets have been advocated since the 1960s as a means of weight loss and to prevent obesity

and its metabolic sequelae (5). However, the long-term safety of such diets has been recently

questioned by increasing evidence of raised cardiovascular risk (6). Accordingly, great excitement

was built by the demonstration that protein-restricted diets (7), or diets with lower concentrations

of single or multiple essential amino acid (e.g., leucine, methionine, or tryptophan; EAAs), were

correlated to improved energy balance and decreased overweight or obesity, in mice and humans

(8,9). In contrast, several studies have also shown the efficacy of central or peripheral

supplementation of a single amino acid (e.g., glycine, leucine, or tryptophan) in modulating energy

metabolism and/or body weight (10,11). However, not all of these findings were confirmed by

others (12).

To investigate these conflicting results, we conducted in-depth analyses on metabolic

effects, in normal-weight and obese mice, caused by acute and chronic consumption of two

customized diets. Specifically, the protein content (i.e., casein) of the SFA diet (10 % fat) and

high-fat diet (HFD, 60 % fat) was substituted with an original formula composed by EAAs (SFA-

EAA and HFD-EAA) (for nutrient compositions see below). The EAA mixture was

stoichiometrically similar to the formula we previously showed to promote mitochondrial

biogenesis in skeletal and cardiac muscles of middle-aged mice and to ameliorate health, especially

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in older adults, when consumed as a dietary supplement with drinking water (13,14). The metabolic effects of SFA-EAA and HFD-EAA diets were compared to those owed to two isocaloric, isolipidic, and isonitrogenous diets, identical to SFA or HFD diet except for casein replacement by the purified amino acid mixture designed on the amino acid profile of casein (SFA-

CAA and HFD-CAA diets), in addition to those induced by a chow diet. In particular, drinking supplementation of the EAA mixture was found to prevent oxidative stress in metabolically active cells (14–16), ameliorating muscle and cognitive performance in diverse animal models and humans (17,18). Moreover, a relevant rejuvenation was observed in the gut microbiota of ageing mice supplemented with the EAA mixture (19). This observation seems to be of relevance because diet composition may affect energy balance also through intestinal microbiota. For example, dietary amino acid intake increases the relative abundance of Bacteroidetes (20), and EAA-derived short-chain fatty acids modulate the overall lipid balance and glucose metabolism (21). Also, the gut microbiota controls adiposity (22) and can activate adaptive thermogenesis in mice (23).

Adaptive thermogenesis refers to the generation of heat by the body in response to external stimuli (e.g., cold temperature, ingestion of high-calorie foods). It includes shivering and non- shivering thermogenesis (NST) The activation of brown adipocytes, in brown adipose tissue

(BAT), and beige adipocytes, in visceral and subcutaneous fat depots, contributes substantially to

NST (24). These thermogenic adipocytes express uncoupling protein 1 (UCP1), a protein that uncouples respiration and leads to energy dissipation in the form of heat. Ablation of brown or beige adipocytes predisposes mice to develop obesity and type 2 diabetes (25,26), as does deletion of the Ucp1 (27). Conversely, increasing the number or activity of thermogenic adipocytes protects against body weight gain and metabolic disease (28). Mechanistic target of rapamycin complex 1 (mTORC1) activation is required for BAT recruitment and metabolic adaptation to cold

(29,30). Additional mechanisms activate thermogenesis in an UCP1-independent manner: the

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glycerol phosphate shuttle, calcium-dependent ATP hydrolysis, and -dependent substrate

cycling (31–33). Also, N-acyl amino acids stimulate uncoupled mitochondrial respiration in white

adipocytes, independently of UCP1, and their systemic administration activates the whole-body

energy expenditure (34).

Here we show that almost complete substitution of the casein in standard rodent diets with

a unique amino acid mixture modulated energy homeostasis in diet-induced obesity or a model of

genetic obesity. Our results reveal multiple thermogenic mechanisms by which designer diets

could be used to combat obesity and type 2 diabetes.

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RESEARCH DESIGN AND METHODS

Animals, diets, and treatments

Male C57BL/6N mice (8 weeks old), from Charles River (Calco, Italy), were weight-matched and fed ad libitum for different periods of time with normal chow diet (CD: V1534-300, Ssniff

Spezialdiäten GmbH, Soest, Germany), SFA diet (20 % protein  namely casein , 70 % carbohydrate, and 10 % fat, half of which was lard (D12450H), HFD diet [20 % protein, 10 % carbohydrate, and 60 % fat (D12492)], or two isocaloric, isolipidic, and isonitrogenous diets, identical to SFA or HFD diet except for protein which was almost completely (93.5 %) replaced by defined free EAAs (SFA-EAA, D14032501 or HFD-EAA, D17073104) or a purified amino acid mixture designed on the amino acid profile of casein (SFA-CAA diet, A17092801, or HFD-

CAA, A20040601) all from Research Diets Inc. (Brogaarden, Gentofte, Denmark) (Supplementary

Table 1).

Body weight and food intake were recorded twice a week in mice housed individually

(except for the survival study); A cohort of C57BL6/N (8 weeks old) and ob/ob (6 weeks old,

C57BL/6J background) male mice were kept and examined at thermoneutrality (30 °C) for the entire treatment period. For norepinephrine (NE) turnover analysis mice fed for 6 weeks with SFA-

CAA and SFA-EAA diets were i.p. treated with -methyl-para-tyrosine (AMPT, Sigma Aldrich) at time 0 (300 mg/kg BW) and after 2 h (150 mg/kg BW). Mice were sacrificed after 4 h and NE content was determined on iBAT with the Noradrenaline High Sensitive ELISA kit (Diagnostika

GMBH, Hamburg, Germany), following manufacturer’s instructions. For selective inhibition of mTORC1, mice were i.p. injected with rapamycin (Rapa, 2.5 mg/kg body weight) or vehicle (Veh,

0.2 % carboxymethylcellulose, 0.25 % Tween 80 in sterile water) (all from Sigma Aldrich), 5 days per week for 6 weeks, starting with diets. All mice used have a C57BL/6N background, that does

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not exhibit defects of insulin release and mitochondrial dysfunction relative to C57BL/6J mice

(35). Body composition were determined using the EchoMRI-100 system (Houston, TX, USA) ,

as previously described (36). Tissue samples for molecular analysis were snap-frozen in liquid

nitrogen and prepared as described below.. All animal procedures were conducted in accordance

with the European Community Guidelines and those of the Italian Ministry of Health, complied

with the National Animal Protection Guidelines.

Caloric absorption and gut motility

At the end of treatments, feces were collected from each mouse, and energy content was measured

by calorimetric bomb analysis (LECO AC-350, Milan, Italy). To monitor gut motility, whole gut

transit time was measured as described (37). As parameters of absorbent capacity and epithelial

morphology, the villi length of jejunum from frozen sections of intestine was determined by the

Nikon Lucia IMAGE (v. 4.61) image analysis software (37).

Glucose homeostasis and biochemical analysis

Glucose (GTT) and pyruvate (PTT) tolerance test were performed after overnight fasting by i.p.

injection of glucose or pyruvic acid (each 1.5 g/kg body weight) (Sigma Aldrich). Insulin tolerance

test (ITT) was performed after 4 h daytime fast, with 0.5 U/kg body weight insulin i.p. injection

(Sigma Aldrich). Glucose levels were measured in tail-vein blood using a Glucometer MyStar

Extra (Sanofi, Milan, Italy) and test strips, at different time points after bolus. Plasma biochemistry

and thyroxine levels were provided by Charles River Laboratories Service (Charles River Clinical

Pathology/Immunology Laboratories, Wilmington, MA, USA). Metabolites and hormones were

measured by commercial kits (Supplementary Table 2). Mean lipid droplet density and area in

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iBAT were measured by the Nikon Lucia IMAGE (v. 4.61) image analysis software and calculated in 25 brown adipocytes for each sample.

Behavioral tests

The social dominance tube test and the elevated plus-maze test were carried out with mice fed with

SFA, SFA-CAA, and SFA-EAA diet for 20 months, as described (38). Spontaneous motor activity was evaluated using an activity cage (Ugo Basile, Varese, Italy) placed in a sound-attenuating room, while an object recognition test was conducted in an open plastic arena.

Indirect calorimetry, locomotor activity, and temperature measurements

Metabolic efficiency was calculated as the ratio between body weight gain and the total energy intake over five days, 2 or 6 weeks. For in vivo indirect calorimetry analysis, mice were transferred in a Phenomaster System (TSE Systems GmbH, Bad Homburg, Germany) 1 week before the study started for acclimatization, followed by three weeks of continued measurements, as previously described (36). Core body temperature was obtained with a rectal probe (Physitemp Instruments

Inc., Clifton, NJ, USA). Shaved back temperature was measured to estimate the level of iBAT thermogenesis, by infrared thermography, with a thermo-electrically cooled Thermocam P25 (Flir

Systems Inc., Wilsonville, OR, USA) (37). In vivo images were captured and analyzed using the

Flir quick report software according to the manufacturer’s specifications.

Mass spectrometry analysis

Plasma samples and iBAT were extracted from mice after night-feeding, and their amino acid content was quantitated using the ACCQ·Tag derivatization reagent provided by Waters

Chromatography Europe BV (Etten-Leur, The Netherlands). All amino acids were acquired in

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positive polarity, in both TOF MS and Product Ion mode, according to the m/z values reported in

Supplementary Table 3.

Fecal microbiota analysis

Fresh feces were collected, immediately frozen and stored. After extraction of total DNA

(FastDNA™ SPIN Kit for Feces, MP Biomedicals, USA), 16S rRNA sequencing analysis was

performed. Alpha and beta-diversity were determined using Quantitative Insight into Microbial

Ecology (QIIME). Data are accessible at the European Nucleotide Archive with the accession

number PRJEB25686.

Primary brown adipocytes

Mouse brown fat precursor cells were enzymatically isolated from iBAT of C57BL/6N male mice

and differentiated in culture for nine days as previously described (39). Primary brown adipocytes

maintained in amino-acid free DMEM (US Biological Life Science, DBA, Milan, Italy) for 16 h,

were pretreated with rapamycin (1.0 nM) or vehicle (DMSO, Sigma Aldrich) for 1 h and

subsequently supplemented for further 24 h with one of two different amino acid combinations,

precisely reproducing the iBAT aminograms resulting from consumption of SFA-CAA or SFA-

EAA diet (Supplementary Table 4). Cellular oxygen consumption rates (OCRs) were determined

using a Seahorse XF24 Extracellular Flux Analyzer (Agilent, Santa Clara, CA, USA). Before

analysis, the culture medium was changed to respiration medium. After basal respiration,

uncoupled and maximal respiration was determined following the addition of oligomycin (1 μM)

and subsequently carbonyl cyanide-4-trifluoromethoxy phenylhydrazone (FCCP, 0.2 μM).

Rotenone (3 μM) was used to abolish mitochondrial respiration. All reagents were purchased from

Sigma-Aldrich.

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Gene expression and mitochondrial physiology

Quantitative RT-PCR reactions were performed as described (32) and run with the iQSybrGreenI

SuperMix (Bio-Rad; Segrate, Italy) on an iCycler iQ Real-Time PCR detection system (Bio-Rad). mtDNA was amplified using primers specific for the mitochondrial cytochrome b gene and normalized to genomic DNA (gDNA). Primers were designed using Primer3 (version 0.4.0) software and are shown in Supplementary Table 5. Immunoblot analysis was performed as described (32). The antibodies (each at 1:1,000 dilution) are reported in Supplementary Table 2.

Citrate synthase activity was measured spectrophotometrically in gastrocnemius as described (32).

Carnitine palmitoyltransferase I (CPT1) activity was measured spectrophotometrically in iBAT mitochondria, as previously described (40). Electron microscopy analysis was conducted in thin sections of iBAT stained with lead citrate and examined with a CM10 transmission electron microscope (Philips, Eindhoven, Netherlands). Mitochondrial OCR was measured in a gas-tight vessel equipped with a Clark-type oxygen electrode (Rank Brothers Ltd., Cambridge, UK).

Briefly, mitochondria from iWAT and iBAT were isolated, and respiration was measured as described (34). Liver and gastrocnemius mitochondria respiration was assessed in 137 mM KCl,

10 mM HEPES pH 7.2, 2.5 mM MgCl2, 0.1% BSA, and 2 mM K2HPO4. iBAT OCR was measured by sequential addition of 2.5 mM malate, 30 μM palmitoyl-CoA plus 5 mM carnitine, 2 mM GDP,

100 M ADP, 0.01 mg/mL oligomycin, and FCCP (500 nM), all from Sigma Aldrich. UCP1- dependent respiration was calculated as the amount of ADP-independent respiration that was inhibited by GDP, as described (24). For iWAT, liver, and gastrocnemius the same substrates, except GDP, were used.

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N-acyl amino acid and acylcarnitine measurements by LC-HRMS

N-acyl amino acids were synthesized and measured, as previously described (34). Targeted

quantitative analysis was carried out on extracted ion chromatograms corresponding to the

molecular ion m/z values [M-H]- for N-acyl amino acid and [M+H]+ for long-chain acylcarnitines

(LCACs). Quantitation of N-acyl amino acid and LCACs was performed by integrating the area

under the peak of each natural compound and normalizing to the corresponding internal standard

area value.

Statistical analyses

Statistical analysis was performed by unpaired Student’s t-test for two-group analysis or one-way

ANOVA with Tukey correction or two-way ANOVA with Bonferroni correction for multiple

group comparisons. Kaplan–Meier survival curves were created with the log-rank test equal to the

Mantel–Haenszel test. A P value < 0.05 was considered statistically significant. Correlations were

determined by nonparametric Spearman correlation test using the computer program GraphPad

Prism (version 6.04).

Data and Resource Availability

The data sets generated and analyzed during the current study are available from the corresponding

author upon reasonable request.

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RESULTS

Effects on body weight, body composition, and adipose tissue

The subacute (6-week) and chronic (80-week) consumption of SFA diet increased body weight and adiposity independently of body length and lean mass, with higher circulating leptin levels compared to mice fed a chow diet (Fig. 1A-D and Supplementary Fig. 1A). Feeding mice with

SFA-EAA diet prevented obesity development, maintaining lower fat mass and circulating leptin levels, without affecting lean mass or the normal growth of mice (Fig. 1A-D and Supplementary

Fig. 1A and B). Notably, after 44 weeks mice fed with SFA-EAA diet weighed less than mice fed with a chow diet, with reduced body weight and adiposity until sacrifice (Fig. 1A and C). In addition to this preventive anti-obesity action, our designer diet exerted a potent therapeutic effect in which mouse body weight dropped after the switch from the SFA diet to the SFA-EAA diet

(Fig. 1A). Body weight of the SFA-fed mice that were switched to the SFA-EAA diet indeed decreased to the level of chow-fed mice in ~2.5 months (Fig. 1A). Notably, the specific EAA substitution for casein entirely prevented the body weight gain, fat mass accumulation, and adiposity also induced by HFD consumption (Supplementary Fig. 2A-C). Similarly, the HFD-EAA diet strongly reduced body weight in already obese mice (Supplementary Fig. 2A, left). By contrast, both CAA-substituted diets were unable to prevent the obesity development or reduce body weight in already obese animals (Fig. 1A-D, Supplementary Fig. 2A-C).

The experiments mentioned above were conducted at room temperature (i.e., at ~ 22 °C), a condition known to cause an increase of food intake and metabolism in small rodents to maintain body temperature (41). Thus, to determine the contribution of environmental thermal stress, we tested the metabolic effect of our engineered diets in C57BL6/N and leptin-deficient ob/ob mice

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at thermoneutrality (41). Our additional experiments confirmed the heathy effects of SFA-EAA

diet under this condition (Supplementary Fig. 2D).

Effects on glucose homeostasis and adipokines

Mice fed with the SFA-EAA diet for either six weeks or 11 months at room temperature had

improved glucose and pyruvate tolerance and insulin-induced glycemia-lowering response

compared to those of mice fed with either the SFA or SFA-CAA diets (Fig. 1F and Supplementary

Fig. 1C-G). Fasting blood glucose and insulin concentrations did not differ among mice fed with

different diets (Supplementary Table 6). These phenotypes were also observed when the SFA-

EAA diet was consumed at 30 °C in C57BL6/N (Fig. 1G) and ob/ob mice (Supplementary Fig.

2E), and in HFD-EAA-fed mice at room temperature (Supplementary Fig. 2F and G). These results

were confirmed by the circulating levels of adiponectin (89 ± 15 % higher, P < 0.05) in mice fed

with the SFA-EAA diet compared to mice fed with the SFA-CAA diet (Fig. 1H). Accordingly to

its inverse association to adiponectin in type 2 diabetes (42), insulin-like growth factor 1 (IGF1)

was reduced in plasma of SFA-EAA-fed mice as compared to SFA-CAA-fed animals (Fig. 1I).

Effects on the whole body and hepatic lipid metabolism

Management of dyslipidemia is a crucial approach to reduce cardiovascular risk in obese and

diabetic patients. Plasma nonesterified fatty acid (NEFA) levels, higher after consumption of the

obesogenic diets (SFA and HFD), were reduced in both SFA-EAA- and HFD-EAA-fed mice, to

values observed in mice on chow diet (Supplementary Fig. 3A). Total cholesterol plasma levels

were increased in HFD-treated mice, whereas circulating triglycerides were similar between

groups (Supplementary Fig. 3B and C). Unlike HFD-CAA, the HFD-EAA diet statistically

prevented cholesterol increase induced by HFD (Supplementary Fig. 3B). These systemic effects

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were mirrored by the ability of both SFA-EAA and HFD-EAA diet to prevent the increased NEFA,

triglyceride and cholesterol levels of the liver in SFA- and HFD-fed mice (Supplementary Fig. 3D-

F). Accordingly, macroscopic liver appearance and liver weight confirmed these results

(Supplementary Fig. 3G and H). Mitochondrial respiration with palmitoyl carnitine as substrate

suggested that EAA substitution promoted fat oxidation in the liver of both SFA- and HFD-fed

mice (Supplementary Fig. 3I). Both CAA-substituted diets were ineffective.

Plasma amino acids

Next, we evaluated the effects of diets on amino acids in plasma. Alanine, glutamine, lysine, and

threonine were higher with both EAA- and CAA-substituted diets than with SFA diet, though

threonine was much more increased in SFA-EAA- than SFA-CAA-fed mice (Supplementary Fig.

4). In addition to threonine, the SFA-EAA diet increased histidine and valine levels over both SFA

and SFA-CAA diet (Supplementary Fig. 4). Glycine and serine levels, which were markedly

augmented in the SFA-CAA mice, with the SFA-EAA diet were similar to those observed in the

SFA-fed mice (Supplementary Fig. 4). Notably, the levels of valine, histidine, and threonine were

inversely correlated to the body weight and adiposity, isoleucine was inversely correlated to the

body weight but not adiposity, while leucine was correlated with neither (Supplementary Table

7). Moreover, tyrosine was positively correlated to fat accumulation, while an inverse correlation

was observed between plasma levels of histidine and the liver weight and liver mitochondrial

respiration (Supplementary Table 7). Of note, under our experimental conditions, no statistical

correlation was observed with insulin sensitivity. SFA-EAA consumption did not affect liver and

kidney function parameters (Supplementary Table 6).

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SFA-EAA diet extends healthy lifespan

Next, we investigated the effects of SFA-EAA diet on mouse life span. The median lifespan for

chow diet-fed mice was 800 days; it was extended from 650 days for SFA mice and 680 days for

SFA-CAA mice to 797 days for SFA-EAA mice (+ 23 % versus SFA and +17 % versus SFA-

CAA) (Fig. 1J). Maximal lifespan was 899 days for chow, 800 days for SFA, 806 days for SFA-

CAA, and 863 days for SFA-EAA mice. While mice fed with the SFA and SFA-CAA diet

exhibited submissive/lethargic behavior after 20 months, mice fed with the SFA-EAA diet

preserved energetic behavior; this was demonstrated by a test for social dominance, with a positive

trend toward less fearful behavior, measured in the elevated plus-maze, and in spontaneous motor

activity (Supplementary Fig. 5A-C). There was no apparent difference in novel object recognition

memory test among dietary groups (Supplementary Fig. 5 D).

Effects on food intake and energy absorption

We then assessed energy input and output. As our mice drank only water and thus did not consume

any calories through drinking, energy input was measured as food intake and energy absorption.

During the first 2 or 5 days of the dietary challenge, mice fed with the EAA-substituted diets

decreased their food intake compared to the respective controls; nevertheless, these differences

vanished at later time points (Fig. 2A and Supplementary Fig. 6A-C ). Similarly, the SFA-CAA-

and HFD-CAA-fed mice decreased their food intake during the first days (Fig. 2A and

Supplementary Fig. 6A). As these mice gained a comparable amount of body weight as SFA- and

HFD-fed mice (Fig. 1B and Supplementary Fig. 3A), SFA-CAA- and HFD-CAA-fed mice served

as pair-fed control to SFA-EAA- or HFD-EAA-fed mice, respectively; henceforward, the SFA-

CAA and HFD-CAA diets were considered the proper control diets.

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Given changes in the ratio of EAAs and nonessential amino acids (NEAAs) in both SFA-

EAA and HFD-EAA versus casein- or CAA-containing diets, we investigated whether some of

the results obtained with the SFA-EAA and HFD-EAA diets are mediated by the detection of a

perturbed amino acid balance by liver or brain. We studied the activity of the critical regulator of

cellular responses under amino acid deficiency sensor, the kinase general control nonderepressible

2 kinase (GCN2). When activated by accumulation of uncharged tRNAs, GCN2 phosphorylates

the  subunit of eukaryotic translation initiation factor 2 (eIF2), inhibiting general protein

synthesis and mitigating the cellular stress (43). We found that the SFA-EAA diet did not change

the Ser51-phosphorylation of eIF2 in both hypothalamus and liver when compared to SFA and

SFA-CAA diets (Supplementary Fig. 6D), suggesting that the effects of EAA-substituted diets

are unlikely caused by detection of a dietary amino acid unbalance.

Next, we assessed the gut size and absorptive capacity of the different dietary regimens.

Both SFA-EAA- and SFA-CAA-fed mice had increased transit time and jejunum villi length

compared to SFA-fed animals (Fig. 2B and C); their percentage of food mass excretion was lower compared to SFA-fed mice, with only a trend to decreased amount of feces excretion (Fig. 2D),

suggesting that both substituted diets may cause malabsorption, albeit they affect differently body

weight. Thus, we investigated whether the SFA-EAA and SFA-CAA exposure led to changes in

calorie uptake, by measuring the fecal caloric content with bomb calorimetry. This analysis

revealed a comparable energy content in the fecal material of mice fed with SFA-EAA or SFA-

CAA diet (Fig. 2E), resulting in similar daily energy excretion in both groups, as compared with

SFA-fed mice (Fig. 2F), also when expressed as a percentage of the food intake. These data

indicate that food malabsorption is unlikely to be the underlying cause of the SFA-EAA diet’s

anti-obesity action.

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Effects on energy expenditure and physical activity

Next, we sought to understand whether energy expenditure was responsible for the observed

phenotypes in mice fed with the different diets. The metabolic efficiency decreased in the SFA-

EAA- and HFD-EAA-fed mice compared to SFA-CAA- and HFD-CAA-fed animals, respectively,

starting at day 5 of treatment (Fig. 3A and Supplementary Fig. 7A), suggesting that the EAA-

designer diets may target energy homeostasis to reverse the obesity-disturbed metabolism

favourably. Oxygen consumption (VO2) and energy expenditure (EE) were measured in the

different dietary groups (Supplementary Table 8). Of note, while no difference was observed in

mice treated for two weeks, VO2 and EE were higher in mice fed with the EAA-substituted diets

for six weeks (Fig. 3B, Supplementary Fig. 7B, and Supplementary Table 8). Locomotor activity,

which is a contributor to total energy expenditure, was normal in mice fed with all of the substituted

diets (Supplementary Table 8). The respiratory exchange ratio (RER) was reduced in SFA-EAA-

and HFD-EAA-fed mice compared to SFA-CAA- and HFD-CAA-fed mice at both time treatment

(Fig. 3C and Supplementary Fig. 7C ). This result is consistent with preferential oxidation of lipid

fuels in SFA-EAA and HFD-EAA mice. Moreover, the increase of core body temperature was

evident only after six weeks of SFA-EAA feeding (Fig. 3D) and was independent of circulating

levels of thyroid hormones, thyroxine (total T4) and thyroid-stimulating hormone (Supplementary

Table 6). Similar results were also observed in HFD-EAA compared to HFD-CAA mice (data not

shown).

Effects on gut microbiota

No significant difference in microbial richness between diets was evident with three different -

diversity estimators (Fig. 4A). The unsupervised multivariate statistical analysis—based on Bray-

Curtis dissimilarity distance (PCoA)—showed that the gut microbiota of SFA-EAA group

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represented a structural shift mainly along with the second principal component (PC2) (Fig. 4B).

In contrast, the gut microbiota of SFA and SFA-CAA groups were not separated (Fig. 4B).

Moreover, the Bray-Curtis dissimilarity index between SFA and SFA-EAA groups was significantly higher compared to each within-group dissimilarity and compared to that between

SFA and SFA-CAA groups (FDR P < 0.001, Fig. 4C). Despite taxon-based analysis revealed small

effects, the SFA-EAA diet consumption promoted an enrichment of Allobaculum and Sutterella

relative abundance compared to SFA (FDR P = 0.029) and SFA-CAA (FDR P = 0.032) diet; in

turn, the SFA diet enriched Lactococcus compared to SFA-CAA diet (FDR P = 0.029) (Fig. 4D

and E and Supplementary Table 9). Furthermore, Allobaculum, Sutterella, and Lactococcus were

shown as the most discriminant bacterial taxa between SFA- and SFA-EAA-fed mice by Random

Forest analysis (Fig. 4F). Notably, Allobaculum relative abundance inversely correlated with the

body weight (Spearman’s r = –0.381, P < 0.05), insulin tolerance (Spearman’s r= –0.529, P <

0.005), and glucose metabolism (Spearman’s r = –0.704, P < 0.01). Similarly, Sutterella inversely

correlated with the insulin tolerance (Spearman’s r = –0.671, P < 0.01) and glucose metabolism

(Spearman’s r = –0.532, P < 0.05), as well as Akkermansia with a reduced body weight

(Spearman’s r = –0.613, P < 0.05).

Effects on thermogenic white adipocytes

To assess the role of adaptive thermogenesis in the action of SFA-EAA diet, we investigated

whether the EAA-substituted diets were able to promote the differentiation of beige adipocytes

within inguinal white adipose tissue (iWAT). profiling after treatment did not

find a statistical increase of markers of beige adipocytes in iWAT, as well as of the thermogenic

UCP1 and PR domain containing 16 (PRDM16), the positive inducer of mitochondrial

biogenesis endothelial nitric oxide synthase (eNOS) (39), or the mitochondrial biogenesis markers

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in mice fed with SFA-EAA diet (Supplementary Fig. 8A-C). Moreover, the expression of genes

involved in lipolysis and fatty acid oxidation remained unchanged, while NEFA levels were

decreased in iWAT of SFA-EAA mice compared to controls (Supplementary Fig. 8D). The

histological analysis confirmed these results, suggesting that browning is unlikely to play an

important role in the metabolic actions of SFA-EAA diet (Supplementary Fig. 8E).

By contrast, we found that the uncoupled respiration was higher in iWAT mitochondria of

wild-type or ob/ob mice fed with the SFA-EAA than SFA-CAA diet at 30 °C—a condition in

which there is no expression of UCP1. This result suggested that the SFA-EAA diet could promote

specific UCP1-independent thermogenic processes by which body temperature is increased (Fig.

5A and B and Supplementary Fig. 9A). Although not directly investigated at a biochemical level,

gene expression analysis suggested that futile cycling, including glycerol phosphate shuttle,

calcium release and reuptake, and creatine-driven substrate cycling (44) remained unchanged after

consuming the SFA-EAA diet (Supplementary Fig. 10A-C ), as well as mitochondrial biogenesis

and function in skeletal muscle (Supplementary Fig. 10D-F). Because N-acyl amino acids

stimulate uncoupled mitochondrial respiration in white adipocytes, independently of UCP1 (34),

we also measured the levels of N-lipidated amino acids in both the plasma and iWAT of SFA-

CAA- and SFA-EAA-fed mice, housed at 30 °C. The circulating and adipose levels of specific

C18:1-amino acids were higher in SFA-EAA than SFA-CAA mice (Fig. 5C). The N-acyl amino

acids were undetectable in the interscapular adipose tissue (iBAT) of mice fed with both diets. The

mRNA levels of Pm20d1 (peptidase M20 domain-containing 1), the secreted by

thermogenic adipose cells that regulates N-acyl amino acids were unaltered in iWAT, iBAT, and

liver of SFA-CAA- and SFA-EAA-fed mice (Fig. 5D).

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Effects on brown adipocytes

Next, by using infrared thermography, we found that after 6 weeks of dietary treatment, iBAT temperature was higher in mice fed with the SFA-EAA diet compared to those fed with the SFA-

CAA diet (Fig. 6A). Interestingly, already at day 5 of treatment, the SFA-EAA diet promoted an uncoupled respiration and the UCP1 protein expression in iBAT mitochondria compared to the control diet (Fig. 6B and C). These effects were also maintained after six weeks of treatment (Fig.

6D and E). Although thermogenic genes type 2 iodothyronine deiodinase (Dio2) and PRDM16 were unaffected, we found an increased expression of nuclear-encoded respiratory complex genes and of mitochondrial DNA amount, in addition to UCP1 and eNOS, in iBAT of mice fed with the

SFA-EAA diet (Supplementary Fig. 11A-C). Moreover, the expression level of the lipolysis- related gene adipose triglyceride lipase (Atgl) was higher in iBAT of mice fed with the SFA-EAA diet than with the SFA-CAA diet (Supplementary Fig. 11D). This was accompanied by a greater preponderance of small lipid droplets (Supplementary Fig. 11E) and a higher level of NEFAs in iBAT of SFA-EAA mice (Supplementary Fig. 11F). These results suggested that fatty acid oxidation was increased in iBAT; accordingly, RER was decreased (Fig. 3C) while carnitine palmitoyltransferase 1 (CPT1) activity was increased in iBAT of mice consuming the SFA-EAA diet (Supplementary Fig. 11G).

The mitochondrial respiratory electron transport chain cytochrome c oxidase subunit IV

(COX IV) and cytochrome c (Cyt c) protein levels were higher in iBAT of SFA-EAA than SFA-

CAA mice (Fig. 6E), suggesting increased mitochondrial biogenesis. In accordance, the density of the mitochondrial cristae was augmented (SFA-EAA diet: 23.64 ± 0.48 m-2 versus SFA-CAA diet: 17.83 ± 0.43 m-2; n = 600 mitochondria for each sample, P < 0.0001), although mitochondrial density was similar in both diets (SFA-EAA: 8.9 over 10 m2 versus SFA-CAA:

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8.1 over 10 m2, ns) and mitochondrial area was reduced (SFA-EAA diet: 0.58 ± 0.017 m2 versus

SFA diet-CAA: 0.71 ± 0.018 m2, P < 0.0001) by the SFA-EAA diet (Fig. 6F). Congruently,

iBAT was activated in obese ob/ob mice (Supplementary Fig. 9B-D). Overall, our findings suggest

that the SFA-EAA diet was able to activate an iBAT thermogenic program, in which utilization

(i.e., oxidation) of fatty acids is increased.

Effects on brown adipocyte activators

Thus, we investigated how the SFA-EAA diet activates thermogenic program in iBAT. Fibroblast

growth factor 21 (FGF21) that promotes sympathetic nerve activity in brown fat, browning of

iWAT, and energy expenditure in diet-induced obese mice (45), was lower in plasma of the SFA-

EAA-fed mice, accordingly to reduced FGF21 gene expression in liver and adipose. These

observations may be of interest, since elevated circulating FGF21 is noted in impaired glucose

tolerance and type 2 diabetes and correlates with muscle and hepatic insulin resistance (46), and

not only with increased thermogenesis and metabolic homeostasis improvement (38).

(Supplementary Fig. 12A and B). Analogously, long-chain acylcarnitines, synthesized in the liver

in response to cold exposure or treatment with 3-adrenergic receptor agonists, and providing fuel

for iBAT thermogenesis (47), were unchanged in plasma of mice fed with either diet, similarly to

the expression of genes involved in their metabolism in the liver, iBAT (except for CrAT), and

muscle (Supplementary Fig. 12C and D).

Also, there was no apparent difference of norepinephrine turnover—a direct neurochemical

measure of sympathetic nervous system (SNS) activity and a proxy of sympathetic innervation

(48)—in iBAT of SFA-EAA mice versus SFA-CAA mice, as well as of tyrosine hydroxylase and

3-adrenoceptor expression, markers of noradrenergic innervation (Fig. 7A and B). Given that

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sympathetic denervation of iBAT—both surgical and chemical—has limitations, especially when used to study the involvement of SNS in systemic effects of chronic treatments, we chose to develop an in vitro system to confirm that the SFA-EAA diet may act directly on brown adipocytes independently of SNS. In particular, we exposed primary brown adipocytes differentiated in culture to one of two different amino acid combinations, precisely reproducing the iBAT aminograms resulting from consumption of either SFA-CAA or SFA-EAA diets (Fig. 7C and

Supplementary Table 4). Notably, the EAA mixture increased both UCP1 mRNA and protein levels and, accordingly, the uncoupled respiration when compared to vehicle-treated cells (Fig.

7D-F). Conversely, the CAA mixture failed to promote either UCP1 expression or oxygen consumption. Together, these findings seem to suggest that the effects of SFA-EAA diet on iBAT depends mainly on a brown adipocyte autonomous process.

Effects on thermogenic signaling

We next aimed to investigate the molecular mechanism of iBAT activation by studying the role of the mechanistic target of rapamycin (mTOR) signaling pathway activated by amino acids and required for brown fat recruitment and metabolic adaptation to cold exposure (29,30). The exposure of primary brown adipocytes in culture to EAA mixture markedly promoted mTOR activation—as shown by the phosphorylation of the mTOR complex 1 (mTORC1)-downstream target ribosomal protein S6 (S6) (Fig. 7G). Despite a partial efficacy of CAA mixture in the mTOR activation, however, this effect was not associated with stimulation of the UCP1-dependent thermogenic program. In turn, rapamycin—the macrolide antibiotic that selectively inhibits mTORC1, fully antagonized the stimulatory effect of EAA mixture on the expression of UCP1 and markers of mitochondrial biogenesis (Fig. 7E). These results were confirmed in vivo, in

C57BL6/N and ob/ob mice (Fig. 8 and Supplementary Fig. 13). Already at day 1 of treatment,

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SFA-EAA diet consistently increased S6 phosphorylation in iBAT (Fig. 8A). Our data showed that

this effect was iBAT-specific, without any phosphorylation of S6 and 4E-BP1 (or eukaryotic

translation initiation factor 4E-binding protein 1) in iWAT and gastrocnemius muscle after 6-

weeks feeding with the SFA-EAA diet (Supplementary Fig. 13B). The systemic administration of

rapamycin entirely blocked oxygen consumption, and S6 and 4E-BP1 phosphorylation in iBAT of

SFA-EAA-fed mice (Fig. 8B and C); these effects were accompanied by a blockade of action of

this diet on core and iBAT temperature, UCP1 expression in brown fat, as well as on body weight

and adiposity, without affecting food intake (Fig. 8D and E and Supplementary Fig. 13C and D).

24 Diabetes Page 26 of 75

DISCUSSION

Collectively, our data reveal that customized diets, in which a precise formula of EAAs substitutes for protein content—without changing calorie content and macronutrient percentage—exert preventive and therapeutic anti-obesity effects, with amelioration of body adiposity, insulin resistance, and fatty liver, beyond promoting a longer and healthier life span. The beneficial effects of either SFA-EAA or HFD-EAA diet on longevity and metabolic homeostasis can be secondary to reduced adiposity, even if the ability of our particular amino-acid combination to play a primary action cannot be excluded.

Although with the EAA-substituted diets certain EAAs (i.e., histidine, isoleucine, leucine, lysine, threonine, and valine) were consumed in a higher amount, while others (i.e., methionine, phenylalanine, and tyrosine) in lower amounts that those recommended for mice by the U.S.

National Research Council (Supplementary Table 10), the circulating levels of EAAs and NEAAs do not seem to exceed tolerable upper and lower levels for individual amino acids. Though few studies report on the side effects of chronic intake of specific amino acid supplements, and there is no adequate dose–response data from human or animal studies to base accepted limits, our results with life-long consumption of markedly changed amino acid combination are conversely reassuring. They show that diets with very restricted—yet not wholly lacking—amounts of

NEAAs (i.e., the SFA-EAA and HFD-EAA diets) are healthy and do not alter animal growth.

Traditionally viewed as amino acids that can be synthesized de novo in adequate amounts by the animal organism to meet the requirements for growth and maintenance, the nutritionally NEAAs are currently considered, in a correct way, necessary for multiple roles in physiology. NEAAs participate in gene expression, cell signalling pathways, antioxidative responses, in addition to regulate neurotransmission and immunity; thus, their non-essentiality has been questioned (42).

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Reduction of food intake and gut energy absorption do not appear to play relevant roles in

the healthy effects of our substituted diets. This conclusion seems to be confirmed by the absence

in mice fed with SFA-EAA or HFD-EAA diets of the physiological adjustments which animals

usually exhibit to maintain energy balance when subjected to a decrease in resource availability

(e.g., increased exploratory activities and intestinal energy absorption (49), or reduced basal

metabolic rate and NST). The NST is indeed increased in SFA-EAA- and HFD-EAA-fed mice.

While browning of iWAT is not induced, a UCP1’s independent uncoupling of respiration is

evident in iWAT of SFA-EAA-fed mice and potentially may promote thermogenesis. Despite the

possibility that the observed increase of lipidated amino-acid levels in iWAT could be responsible

for this higher uncoupled respiration, their relative contribution to the overall phenotype of SFA-

EAA (and also of HFD-EAA) mice remains to be addressed. Most significantly, the SFA-EAA

and HFD-EAA diets promote a marked iBAT activation in both normal weight and obese mice,

irrespective of the environmental temperature. FGF21, acylcarnitines, NE, and thyroid hormones,

well-known activators of brown fat (38,40), are not responsible for the beneficial effects of the

SFA-EAA diet. Similarly, other thermogenic mechanisms, including the UCP1-independent futile

cycling and muscle mitochondrial biogenesis, are not promoted by the SFA-EAA diet.

Conversely, iBAT thermogenesis could be directly activated by the specific combination

of amino acids in this tissue. The EAA mixture, unlike CAA mixture—that were designed on the

profiles of amino acids identified in iBAT of SFA-EAA- and SFA-CAA-fed mice (iBAT

aminograms), respectively—promotes a thermogenic program in cultured brown adipocytes,

through activation of mTORC1 signaling. Notably, the iBAT aminograms differ between mice fed

with the two dietary regimens: arginine, isoleucine, leucine, proline, threonine, and valine are

statistically higher in SFA-EAA- then in SFA-CAA-fed mice while, on the contrary, glycine,

lysine, and serine are lower. From the literature, isoleucine, leucine, and valine (namely, the

26 Diabetes Page 28 of 75

branched-chain amino acids) supplementation ameliorates some metabolic dysfunction caused by obesity or diabetes, without impairing glucose metabolism, in some studies (50), but not in others

(51,52). Arginine, as a substrate of nitric oxide synthase, produces nitric oxide (NO) for signaling purposes—including mitochondrial biogenesis and thermogenic program in iBAT (39). Increasing evidence has shown that dietary supplementation of arginine can effectively improve BAT thermogenesis via the mTOR signaling pathway (53), with reduced obesity and diabetes, in addition to improved obesity-linked dyslipidemia and blood hypertension in mammals, including humans (54). Similarly, proline supplementation improves NO bioavailability and counteracts the blood pressure (55). By contrast, the SFA-EAA diet reduced the NEAA glycine levels in iBAT compared to SFA-CAA diet, and accordingly, the glycine precursor threonine was increased. Of note, neuronal glycine has been found to inhibit sympathetic activation of BAT (56), and although circulating glycine is low in obese subjects, and its supplementation was proposed as a treatment of obesity (57), no clear role has been found in brown fat. Also for serine, which was similarly reduced in SFA-EAA mice, further studies are needed to confirm and extend our findings.

Additionally, we observed some interesting differences in microbiota composition. There was an enrichment of Allobaculum and Sutterella in the gut of mice fed with SFA-EAA diet compared to SFA and SFA-CAA diet, which might play some role in light of the inverse correlation we observed with body weight and insulin resistance, in agreement with previous observations (58). Several studies have demonstrated that the HFD reduces the relative abundance of both taxa in the gut of mice, while multiple dietary interventions, such as prebiotics, probiotics, and berberine—that ameliorate insulin resistance and adipose inflammation—promote the relative abundance of both Allobaculum and Sutterella (59,60). Again, mounting evidence suggests that gut microbiota can stimulate brown fat thermogenesis in mice (29,54,55). Further studies,

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therefore, are warranted to establish metabolic links between gut bacteria such as Allobaculum and

Sutterella and the iBAT activation by our diets.

Similarly, additional work is necessary to understand whether the pro-longevity effects of

SFA-EAA are simply due to the reduced adiposity or to a direct action of our peculiar amino-acid

combination. Remarkably, reducing NEAA concentration has been found to promote replicative

lifespan extension in yeast (61); as in turn increasing EAA concentration prolongs the lifespan,

not only in yeast, but also in Caenorhabditis elegans and, most importantly, in mammals

(14,62,63). The increased chronological life span extension of yeast promoted by EAA

supplementation was found to be accompanied by lower oxidative damage and notably by the

activation, not inhibition as often reported with other life span prolonging methods, of TOR

signaling pathway (61). Yang et al. (19) have moreover reported that an amino acid formula

similar to the amino acid combination in our EAA-substituted diets, slowed the age-related

changes of gut microbiota, without affecting body weight, suggesting that the dietary

supplementation might promote healthy aging independently of its effects on adiposity.

Since our EAA-substituted diets are deficient of some EAA compared to controls, one

could claim that detection of amino acid unbalance or, alternately, a restriction of specific amino

acids may be relevant intermediaries to the beneficial effects of both SFA-EAA and HFD-EAA

diets. These possibilities were proposed to interpret previous findings. For example, mice fed with

diets lacking the single EAA tryptophan had increased resistance to surgical stress, as well as

improved longevity, metabolic fitness and stress resistance (64). However, detection of dietary

amino acid imbalance seems improbable in our case since there is no apparent change of the GCN2

activity, the critical regulator of cellular responses under amino acid deficiency or imbalance, in

both hypothalamus and liver of mice under the different dietary regimens. Moreover, previous

studies have shown that diets deficient in EAAs can influence food intake and increase FGF21

28 Diabetes Page 30 of 75

levels (65). Consuming SFA-EAA diet, conversely, does not change feeding and decreases circulating and hepatic FGF21 levels. Also, feeding rodents survival-promoting methionine- restricted diets comprising purified amino acids as the sole source of nitrogenous content causes some of the phenotypes described here, including protection from weight gain (mostly in adiposity), improved glucose homeostasis, increased energy expenditure, and activation of iBAT

(66). However, the abundance of cysteine in SFA-EAA and HFD-EAA diets rules out methionine- restriction as the key mechanism behind the present findings.

In summary, the present study indicates that specific manipulation of dietary amino acids prevents and reverses obesity in mice through multiple modes to stimulate thermogenesis.

Reasonably, taken separately each other, these multiple modes would be unable to explain the overall observed results. If extended to humans, such dietary manipulation could potentially have a positive impact on metabolic health favoring the prevention and treatment of obesity and type 2 diabetes regardless of calorie consumption.

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Acknowledgments. The authors thank M. Rossato (Department of Medicine, University of Padua,

Padua, Italy) for experiments with the Flir camera; Mariaelvina Sala and Daniela Braida

(Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan,

Italy) for behavioral experiments; Annapaola Andolfo and Cinzia Magagnotti (Protein

Microsequencing Facility, San Raffaele Scientific Institute, Milan, Italy) for mass spectrometry

analysis, and Alessandra Micheletti (Department of Environmental Sciences and Politics,

University of Milan, Milan, Italy) and Marika Vezzoli (Department of Molecular and Translational

Medicine, Brescia University, Brescia, Italy) for statistical analysis.

Funding. This work was supported by Fondazione Umberto Veronesi to C.R., University of Milan

to F.R. (Research Fellowship grant 1280/2016), Professional Dietetics (Milan, Italy) to E.N.

(support to laboratory), Cariplo Foundation to E.N. and to A.V. (grant 2016-1006), and Louis-

Jeantet Foundation grant to R.C.

Duality of Interests. No potential conflicts of interest relevant to this article were reported.

Author Contributions. C.R. and M.R. designed and performed molecular biology, metabolic,

oxygen consumption, and animal experiments; P.C., C.G., D.L., and R.P.C. designed and

performed experiments in animals; V.G., L.Tenori, and C.L. designed and analyzed experiments

on metabolites; R.M.I., F.Bontems, and R.C. designed, performed, and analyzed indirect

calorimetry experiments; A.C., F.P. and V.D. designed, performed, and analyzed liquid

chromatography-mass spectroscopy experiments for N-acyl-amino acid and acylcarnitine

measurements; E.M. synthesized N-acyl-amino acids; L.Tedesco and F.R. performed in vitro

experiments and qRT-PCR analysis; A.P. and I.D. performed experiments at thermoneutrality;

30 Diabetes Page 32 of 75

L.P. designed, performed, and analyzed behavioral experiments; L.R., M.U.S., and K.T. performed microbiota analysis; I.S. and S.Cinti designed, performed, and analyzed electron microscopy and histological experiments; F.B., M.O.C, G.C., R.C., S.Collins, and A.V. assisted with experimental design and co-edited the manuscript; E.N. conceived the study, designed experiments, and provided advice; C.R., M.R., and E.N. wrote the manuscript with suggestions from all authors, all of whom read and approved the final version. E.N. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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Figure Legends

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 lifespan.

A: Body weight of mice fed with 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 SFA were switched to either SFA-EAA diet (SFA > SFA-EAA) or SFA-CAA diet (SFA

> 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 time points at room temperature (n = 5 mice

per group). E: Body weight of mice at thermoneutrality (30 °C) (n = 10 mice per group). F and

G: Glucose tolerance tests (GTT) 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 six weeks. H

and I: Plasma levels of adiponectin (H) and IGF-1 (I) in mice fed with SFA-CAA and SFA-EAA

diet for six 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 m. D: energy excretion: the mean amount of feces excreted per mouse in 24 h

32 Diabetes Page 34 of 75

(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 SFA, SFA-CAA, and SFA-EAA diets for six 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 gain to the energy intake ratio (i.e., total food consumed during

five days or 2 or 6 weeks). B and C: Energy expenditure (B) and respiratory exchange ratio (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 alpha-diversity calculated on the number of observed OTUs. B: Principal coordinates analysis (PCoA) of bacterial beta-diversity based on the Bray-Curtis dissimilarity index. Each symbol represents a single sample of feces after six weeks of treatment. C: Box-and-whisker plot

of intercommunity beta-diversity determined by 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 box-and-whisker plots, boxes

show median, first and third quartiles. The whiskers extend from the quartiles to the last data point

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within 1.5× IQR, 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 — SFA-EAA diet promotes uncoupled respiration in inguinal WAT (iWAT) without

browning stimulation. A: Uncoupled (i.e., with oligomycin) and maximal (i.e., with FCCP)

oxygen consumption rates (OCR) 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 chow diet at room temperature was used

as control. C: Thermogenic N-acyl amino acids in plasma (left) and iWAT (right) (n = 4-5 mice

per group). D: Relative mRNA levels of Pm20d1 gene in different tissues (n = 5 mice per group).

A-D, mice were fed with SFA-CAA and SFA-EAA diet for six 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 thermographic Flir camera in mice fed with SFA-CAA and SFA-EAA diet for six

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) oxygen consumption rates (OCR) 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 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, COX IV, Cyt c,

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and GAPDH protein levels in iBAT. One experiment representative of three reproducible ones (n

= 4 mice per group). F: Electron microscopy analysis of iBAT. Scale bar, 0.5 m (n = 2 mice per

group). Mice in D-F were fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature. All data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 vs. SFA-CAA diet.

UCP1, uncoupling protein 1; COX-IV, cytochrome c oxidase subunit IV; Cyt c, cytochrome c;

GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 7 — SFA-EAA diet activates brown adipocytes. A: Norepinephrine (NE) turnover (NETO) in iBAT of mice fed with SFA-CAA and SFA-EAA diet for six 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 SFA-CAA

and SFA-EAA diet for six weeks. C: Scheme of in vitro experiment in primary brown adipocytes

isolated from mice fed with chow diet. After 16 h incubation in amino acid-free medium, brown

adipocytes were pretreated with rapamycin (Rapa, 1.0 nM) or Veh (DMSO) for one h. Then, the

cells were supplemented with Veh (PBS) or CAAm or EAAm, specifically reproducing the iBAT

aminograms resulting from consumption of either 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. E and G: Western blot analysis of UCP1

and GAPDH (E), and (Ser 235/236) phosphorylated S6 and S6 (G) protein levels in differentiated

brown adipocytes, treated as in C. One immunoblot experiment representative of three reproducible ones. F: Oxygen consumption rates (OCR) of differentiated brown adipocytes,

treated as in C. OCR was measured with Seahorse XF24 Extracellular Flux Analyzer (n = 3

readings in quadruplicate/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.

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EAAm + Rapa. TH, tyrosine hydroxylase; 3AR, 3-adrenergic receptor; CAAm, casein amino-

acid mixture; EAAm, essential amino-acid mixture; UCP1, uncoupling protein 1; GAPDH,

glyceraldehyde 3-phosphate dehydrogenase; S6, ribosomal protein S6.

Figure 8 — mTORC1 signaling contributes to iBAT thermogenesis induced by SFA-EAA diet.

A: Western blot analysis of (Ser 235/236) phosphorylated S6 and S6 protein levels in iBAT of

mice fed with SFA-CAA and SFA-EAA diet for different time intervals at room temperature. One

immunoblot experiment representative of three reproducible ones (n = 3-5 mice per group). B:

Western blot analysis of the mTORC1 pathway in iBAT of mice fed with SFA-CAA and SFA-

EAA diet, with vehicle (Veh) or rapamycin (Rapa) (n = 4 mice per group). C: UCP1-dependent

(ΔGDP) and maximal (FCCP) oxygen consumption rates (OCR) 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 SFA-CAA and SFA-EAA diet for six weeks at

room temperature, with or without rapamycin (i.p. 2.5 mg/kg body weight) delivered in 200 μl, 5

days per week for six 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. S6, ribosomal protein S6; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1

phosphorylation; UCP1, uncoupling protein 1.

36 Diabetes Page 38 of 75

References

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Online Supplemental Materials

Manipulation of dietary amino acids prevents and reverses obesity in mice through multiple mechanisms that modulate energy homeostasis Chiara Ruocco, Maurizio Ragni, Fabio Rossi, Pierluigi Carullo, Veronica Ghini, Fabiana Piscitelli, Adele Cutignano, Emiliano Manzo, Rafael Maciel Ioris, Franck Bontems, Laura Tedesco, Carolina Greco, Annachiara Pino, Ilenia Severi, Dianxin Liu, Ryan P. Ceddia, Luisa Ponzoni, Leonardo Tenori, Lisa Rizzetto, Matthias Scholz, Kieran Tuohy, Francesco Bifari, Vincenzo Di Marzo, Claudio Luchinat, Michele O. Carruba, Saverio Cinti, Ilaria Decimo, Gianluigi Condorelli, Roberto Coppari, Sheila Collins, Alessandra Valerio, and Enzo Nisoli Page 51 of 75 Diabetes

Supplementary Figure 1 — EAA unlike CAA substitution reduces fat accumulation maintaining the healthy effect on glucose homeostasis also after a long period of treatment. A: Body length as nasal-to-anal distance (n = 8 mice per group) of mice fed with different dietary regimens for 6 and 80 weeks. B : Weight of different fat pads expressed as mg over whole-body weight C-G: Insulin tolerance tests (ITT) (C and F), pyruvate tolerance tests (PTT) (D and G), and glucose tolerance tests (E) in mice fed with chow, SFA, SFA-CAA, and SFA-EAA diets for 6 weeks or 11 months. iBAT, interscapular brown fat; iWAT, inguinal fat; eWAT, epididymal fat; mWAT, mesenteric fat. All data 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.

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Supplementary Figure 2 — EAA-substituted diet prevents and reverses obesity and glucose imbalance induced either by consumption of HFD diet or in ob/ob mice. A: (Left) Body weight of mice fed with chow, HFD, HFD-EAA, and HFD-CAA diet at room temperature (n = 7-10 mice per group). After nine weeks (dashed line), mice fed with HFD were switched to either HFD-EAA diet (HFD > HFD-EAA) or HFD-CAA diet (HFD > SFA-CAA) (n = 5 mice per group). (Right) Body weight of mice fed with chow, HFD, HFD-EAA, and HFD-CAA diet for 18 weeks (n = 5 mice per group). B: Body composition: fat mass (left) and lean mass (right). C: Weight of different fat pads expressed as mg over whole-body weight. Mice in B and C were fed with HFD, HFD-EAA, and HFD-CAA for six weeks (n = 5-6 mice per group). D and E: Body weight (D) and glucose tolerance tests (GTT) (E) in ob/ob mice, housed at thermoneutrality (30 °C) and fed with SFA, SFA-EAA, and SFA-CAA for six weeks (n = 6 mice per group). F and G: Glucose homeostasis in HFD groups. GTT (F) and insulin tolerance tests (ITT) in mice fed with chow, HFD, HFD-EAA, and HFD-CAA diets for six weeks (n = 5 mice per group). All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. SFA or HFD diet; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. SFA-CAA diet and HFD-CAA; §P < 0.05, §§P < 0.01, and §§§P < 0.001 vs. chow diet.

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Supplementary Figure 3 — Effects on lipid homeostasis. A-C: Plasma concentrations of nonesterified fatty acids (NEFAs) (nmol/l) (A), cholesterol (mg/dl) (B), and triglycerides (mg/dl) (C). D-F: Hepatic content of NEFAs (nmol/mg protein) (D), total cholesterol (nmol/mg tissue) (E), and triglycerides (nmol/mg tissue) (F). G: Representative macroscopic pictures of the liver. H: Liver weight (mg/BW). I: Oxygen consumption rates (OCR) were normalized to mitochondrial protein content. Mitochondria were isolated from liver of mice fed with different dietary regimens for 6 weeks at room temperature. Data are presented as means ± SEM (n = 5-8 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. SFA-fed mice; §P < 0.05, §§P < 0.01, and §§§P < 0.001 vs. chow diet-fed mice; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. SFA-CAA-fed mice; ¶¶P < 0.01 and ¶¶¶P < 0.001 vs. HFD-fed mice; †P < 0.05 and †††P < 0.001 vs. HFD-CAA-fed mice.

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Supplementary Figure 4 — Plasma amino acid profile. A-R: Amino acid concentrations in plasma samples of mice fed with SFA, SFA-EAA, and SFA-CAA diet for six weeks at room temperature. Box plots, with center hinge representing the mean, and the upper and lower hinges representing the first and third quartiles; whiskers represent 1.5 times the interquartile range. All data are presented as mean ± SEM (n = 5 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. SFA diet; #P < 0.05, ##P < 0.01, and ###P < 0.01 vs. SFA-CAA.

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Supplementary Figure 5 — SFA-EAA diet consumption preserved energetic behavior. Mice fed with SFA, SFA-CAA, and SFA-EAA for 20 months at room temperature. A: Energetic behavior, as demonstrated by the percentage of winning in the tube test for social dominance, was preserved in mice fed with SFA-EAA compared to SFA and SFA-CAA diets. B: Anxiety-like behaviour, C: Motor activity and D: Episodic memory were similar in all dietary groups. Mice were subjected to familiarization (T1) and novel object recognition (T2). The novel object recognition task was performed using a delay time of 120 min. The performance was evaluated by calculating a discrimination index (N−F / N+F), where N = time spent exploring the new object during T2, F = time spent exploring the familiar object during T2. Mean ± SEM are reported (n = 5 mice per group). ***P < 0.001 vs. SFA- and SFA-CAA-fed mice (Student t-test) and ##P < 0.01 vs. SFA-and SFA- CAA-fed mice (Chi-square test).

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Supplementary Figure 6 — Both EAA and CAA substitution does not affect food intake and stress response pathway linked to amino acid balance in both SFA and HFD diet. A-D: Food intake was measured twice a week in different models of obesity (n = 5-10 mice per group). E: Western blot analysis of (Ser51) phosphorylation of  subunit of eukaryotic translation initiation factor 2 (eIF2)—a marker of kinase general control nonderepressible 2 (GCN2) kinase activity, the critical regulator of cellular responses under amino acid deficiency sensor—in hypothalamus and liver of mice fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature. One experiment representative of three reproducible ones is shown. CTRL+, internal positive control (i.e., amino acid starved HeLa cells). All data are presented as mean ± SEM.

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Supplementary Figure 7 — Energy metabolism in mice on HFD diets. A: Metabolic efficiency was calculated as the ratio between the body weight gain and the energy intake (i.e., total food consumed during five days or 2 weeks or 6 weeks). B and C: Energy expenditure (EE) (B) and respiratory exchange ratio (RER) (C) during one 24 h cycle. Mice in B and C were fed with HFD-CAA and HFD- EAA diets in two separate experiments for 2 or 6 weeks. All data are presented as mean ± SEM. *P < 0.05, and ***P < 0.001 vs. HFD-CAA diet.

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Supplementary Figure 8 — SFA-EAA diet does not promote browning of iWAT. A: Relative mRNA levels of UCP1 and browning genes. B: Relative mRNA levels of eNOS and mitochondrial biogenesis genes. C: Mitochondrial DNA amount. D: Relative mRNA levels of lipolysis and fatty acid oxidation genes (left) and non-esterified fatty acid (NEFA) levels (right). The experiments were repeated at least three times. E: Hematoxylin and eosin staining of semithin sections (3 m) of iWAT. Scale bar, 50 m. All data are presented as mean ± SEM (n = 5 mice per group). *P < 0.05 vs. SFA- CAA diet.

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Supplementary Figure 9 — SFA-EAA diet confirms its thermogenic effects on iWAT and iBAT of ob/ob mice. A: Uncoupled (i.e., with oligomycin) and maximal (i.e., with FCCP) oxygen consumption rates (OCR) in mitochondria isolated from iWAT; respiration was normalized to mitochondrial protein content (n = 5 mice per group). B: iBAT temperature measurement with Flir camera (n = 6 mice per group). C: Western blot analysis of UCP1 and vinculin protein expression in iBAT. One experiment representative of three reproducible ones (n = 5 mice per group). D: UCP-1-dependent (i.e., ΔGDP) and maximal (i.e., with FCCP) OCR in iBAT mitochondria (n = 6 mice per group); respiration was normalized to mitochondrial protein content. ob/ob mice in A-D were fed with SFA- CAA and SFA-EAA diet for six weeks at thermoneutrality. All data are presented as mean ± SEM. *P < 0.05 and ***P < 0.001 vs. SFA-CAA diet.

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Supplementary Figure 10 — SFA-EAA diet does not promote futile cycle activation or mitochondrial biogenesis stimulation in adipose and muscle. A-C: Relative mRNA levels of futile cycles genes, including glycerol phosphate shuttle in iWAT and gastrocnemius (A), creatine-driven substrate cycle in iWAT and iBAT (B), and calcium release and reuptake cycle in gastrocnemius (ATPase Sarcoplasmic/Endoplasmic Reticulum Calcium Transporting 1 - ATP2A1) (C) of mice fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature. D-F: Mitochondrial biogenesis and function in gastrocnemius muscle of mice fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature. Relative mRNA levels of mitochondrial biogenesis genes (D), citrate synthase activity (normalized to protein content) (E), and oxygen consumption rates (OCR) (normalized to mitochondrial protein content) (F). All data are presented as mean ± SEM (n = 5-6 mice per group).

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Supplementary Figure 11 — SFA-EAA diet increases expression of genes involved in mitochondrial biogenesis, thermogenesis, lipolysis, and fatty acid oxidation in iBAT of mice fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature. A: Relative mRNA levels of thermogenic genes. B: Relative mRNA levels of mitochondrial biogenesis genes. C: Mitochondrial DNA (mtDNA) amount. D: Relative mRNA levels of lipolysis and fatty acid oxidation (FAO). The experiments were repeated at least three times. E: Lipid droplets in iBAT slices (toluidine blue staining) and lipid droplet area and density. Scale bar, 25 m. F: Non-esterified free fatty acid (NEFA) levels. G: Carnitine palmitoyltransferase I (CPT1) activity in iBAT mitochondria. All data are presented as mean ± SEM (n = 5-6 mice per group). *P < 0.05 and **P < 0.01 vs. SFA-CAA diet.

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Supplementary Figure 12 — SFA-EAA diet does not induce production of thermogenic FGF21 and long-chain acylcarnitines. A: Relative mRNA levels of Fgf21 gene in different tissues of mice fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature. B: Plasma FGF21 concentrations in mice fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature. C: Serum long-chain acylcarnitine levels of mice fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature analyzed using tandem mass spectrometry. D: Relative mRNA levels of genes implicated in acylcarnitine synthesis in different tissues of mice fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature. The experiments were repeated at least three times. CrAT, carnitine acetyltransferase; Cact, carnitine-acylcarnitine translocase; CPT1a, carnitine palmitoyltransferase 1a; CPT1b, carnitine palmitoyltransferase 1b; Octn2, organic cation transporter type 2. All data are presented as mean ± SEM (n = 5 mice per group). *P < 0.05 and **P < 0.01 vs. SFA-CAA diet.

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Supplementary Figure 13 — mTORC1 signaling contributes to iBAT thermogenesis induced by SFA-EAA diet under different experimental settings. A: Western blot analysis of (Ser 235/236) phosphorylated S6 and S6 protein levels in iBAT of ob/ob mice fed with SFA-CAA and SFA-EAA diet for six weeks at thermoneutrality. One immunoblot experiment representative of three reproducible ones (n = 3-5 mice per group). B: Western blot analysis of mTORC1 pathway in iBAT, iWAT, and muscle of mice fed with SFA, SFA-CAA, and SFA-EAA diet for six weeks at room temperature (n = 4 mice per group). C: Body weight (left) and fat depots (expressed as mg over whole-body weight) (right). D: Food intake (expressed as kcal per mouse per day). Mice in C and D were fed with SFA-CAA and SFA-EAA diet for six weeks at room temperature, with or without Rapa (i.p. 2.5 mg/kg body weight) delivered in 200 μl, 5 days per week for six weeks, starting with diets (n = 5-6 mice per group). All data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 vs. Veh- treated mice fed with SFA-CAA diet.

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Supplementary Table 1 — Diet compositions

Dietary interventions % kcal from SFA SFA-EAA SFA-CAA HFD HFD-EAA HFD-CAA Protein 20 20 20 20 20 20 Carbohydrate 70 70 70 20 20 20 Fat 10 10 10 60 60 60 kcal/g 3.8 3.8 3.8 5.2 5.2 5.2 Amino acid sources SFA SFA-EAA SFA-CAA HFD HFD-EAA HFD-CAA Amino acids (g) caseina caseinb EAAmc caseinb CAAmd caseina caseinb EAAmc caseinb CAAmd Leucine 18 1.17 58.6 1.17 16.8 18 1.17 58.6 1.17 16.8 Valine 10.6 0.69 29.22 0.69 9.91 10.6 0.69 29.22 0.69 9.91 Lysine 15 0.98 30.34 0.98 14.03 15 0.98 30.34 0.98 14.03 Isoleucine 8.6 0.56 29.22 0.56 8.04 8.6 0.56 29.22 0.56 8.04 Threonine 8.2 0.53 16.48 0.53 7.67 8.2 0.53 16.48 0.53 7.67 Histidine 5.2 0.34 7.12 0.34 4.86 5.2 0.34 7.12 0.34 4.86 Cysteine 3.4 0.09 7.12 0.09 1.31 3.4 0.09 7.12 0.09 1.31 /cystinee Phenylalanine 9.6 0.62 4.68 0.62 8.98 9.6 0.62 4.68 0.62 8.98 Methionine 5.8 0.38 2.44 0.38 5.42 5.8 0.38 2.44 0.38 5.42 Tyrosine 10.4 0.68 1.31 0.68 9.72 10.4 0.68 1.31 0.68 9.72 Tryptophan 2.4 0.17 0.94 0.17 2.24 2.4 0.17 0.94 0.17 2.24 Alanine 5.8 0.4 - 0.4 5.42 5.8 0.4 - 0.4 5.42 Arginine 6.8 0.44 - 0.44 6.36 6.8 0.44 - 0.44 6.36 Aspartic acid 13.8 0.9 - 0.9 12.9 13.8 0.9 - 0.9 12.9 Glutamic acid 43.4 2.82 - 2.82 40.58 43.4 2.82 - 2.82 40.58 Glycine 3.4 0.22 - 0.22 3.18 3.4 0.22 - 0.22 3.18 Proline 20.2 1.31 - 1.31 18.89 20.2 1.31 - 1.31 18.89 Serine 11.4 0.74 - 0.74 10.66 11.4 0.74 - 0.74 10.66 Caloric amino acid intakef Δ Δ caloric caloric Δ caloric HFD- Δ caloric kcal/day SFA SFA-EAA intake SFA-CAA intake HFD HFD-EAA intake CAA intake Leucine 1.041 3.425 2.384 1.042 0.001 1.027 3.326 2.299 1.029 0.003 Valine 0.585 1.709 1.125 0.620 0.035 0.577 1.660 1.084 0.612 0.036 Lysine 0.864 1.785 0.921 0.871 0.007 0.852 1.733 0.882 0.860 0.009 Isoleucine 0.497 1.704 1.207 0.498 0.001 0.490 1.655 1.165 0.492 0.002 Threonine 0.474 0.973 0.500 0.481 0.008 0.467 0.945 0.478 0.476 0.009 Histidine 0.296 0.419 0.123 0.296 0.000 0.292 0.407 0.115 0.292 0.000 Cysteine 0.192 0.405 0.213 0.189 0.393 0.204 0.077 -0.113 /cystinee 0.078 -0.114 Phenylalanine 0.563 0.307 -0.256 0.560 -0.003 0.555 0.299 -0.257 0.553 -0.002 Methionine 0.348 0.152 -0.196 0.329 -0.019 0.343 0.148 -0.195 0.325 -0.018 Tyrosine 0.591 0.110 -0.481 0.606 0.015 0.582 0.106 -0.476 0.598 0.016 Tryptophan 0.147 0.067 -0.080 0.141 -0.006 0.145 0.065 -0.080 0.140 -0.006 Alanine 0.329 - - 0.343 0.013 0.325 - - 0.338 0.014 Arginine 0.386 - - 0.389 0.003 0.380 - - 0.384 0.004 Aspartic acid 0.781 - - 0.807 0.026 0.770 - - 0.797 0.027 Glutamic acid 2.205 - - 2.499 0.294 2.174 - - 2.469 0.294 Glycine 0.192 - - 0.201 0.009 0.189 - - 0.199 0.010 Proline 1.147 - - 1.180 0.033 1.131 - - 1.165 0.035 Serine 0.647 - - 0.667 0.020 0.638 - - 0.659 0.021

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aSingle amino acid amount in 200 g of casein (composition provided by Research Diets); bsingle amino acid amount in 13 g of casein; csingle amino acid amount of EAAm [the specific amino acid formula is stoichiometrically similar to a mixture we previously found to promote mitochondrial biogenesis in cardiac and skeletal muscle when administered via drinking water (32)]; and dCAAm, purified amino acid mixture designed on the amino acid profile of casein. The amount of EAA and CAA mixture in the substituted diets was calculated as 93.5 % casein content (187 g out 200 g); ereported amount is equal to the cysteine/cystine ratio typically present in 200 g casein (i.e., 1.4 g) added with further 3.0 g cystine; fcaloric amino acid intakes (kcal/day) were determined on average kcal/day consumed by mice fed with SFA (14.44 ± 0.61 kcal/day), SFA-EAA (14.29 ± 0.45 kcal/day), SFA-CAA (14.54 ± 0.61 kcal/day), HFD (14.24 ± 0.45 kcal/day), HFD-EAA (13.88 ± 0.73 kcal/day), and HFD-CAA (14.36 ± 0.33 kcal/day). The optimal amino acid amounts in the diets were assessed by preliminary experiments, in which different percentages of casein were substituted with the EAA mixture.

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Supplementary Table 2 — Antibodies and assay kits

REAGENT SOURCE CAT. # SAMPLE TYPE Assay kits Leptin (mouse) ELISA Kit Enzo Life Sciences ADI-900-019A Plasma Mouse Adiponectin ELISA Kit Abcam ab108785 Plasma Mouse/Rat FGF-21 Quantikine R&D Systems MF2100 Plasma, iWAT, iBAT, liver ELISA Kit TSH (Rodent) ELISA Kit Abnova KA2336 Plasma Free Fatty Acid Quantification BioVision K612-100 Plasma, iWAT, iBAT, liver Colorimetric/Fluorometric Kit Mouse Ultrasensitive Insulin Alpco 80-INSMSU-E01 Plasma ELISA kit Mouse IGF-1 ELISA Kit Thermo Fisher Scientific EMIGF1 Plasma Cholesterol Assay Kit Abcam ab65390 Plasma, Liver Triglyceride Assay Kit Abcam ab65336 Plasma, Liver Antibodies iWAT, iBAT, gastrocnemius, anti-p-S6 (Ser235/236) Cell Signaling 2211 primary brown adipocytes iWAT, iBAT, gastrocnemius, anti-S6 Cell Signaling 2217 primary brown adipocytes anti-p-4EBP1 (Thr37/46) Cell Signaling 2855 iWAT, iBAT, gastrocnemius anti-4EBP1 Cell Signaling 9644 iWAT, iBAT, gastrocnemius anti p-eIF2α Cell Signaling 3398 Hypotalamus, Liver anti eIF2α Cell Signaling 5324 Hypotalamus, Liver anti-COX IV Cell Signaling 4844 iBAT anti-Cyt c Cell Signaling 4280 iBAT iWAT, iBAT, anti-UCP1 Abcam ab23841 primary brown adipocytes iBAT, anti-GAPDH Cell Signaling 2118 primary brown adipocytes anti-HSP60 Cell Signaling 4870 iWAT anti-vinculin Sigma Aldrich V9131 iBAT

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Supplementary Table 3 — Mass spectrometry parameters

Amino acid RT (min) m/z Calibration curve r Alanine 2.86 260.1 y = 13573.47x 0.99 Glycine 2.51 246.1 y = 11700.40x 0.99 Isoleucine 5.88 302.1 y = 11676.90x 0.97 Phenylalanine 6.53 336.1 y = 6553.14x 0.96 Valine 4.06 288.1 y = 16128.62x 0.97 Threonine 2.74 290.1 y = 8556.89x 0.99 Serine 2.46 276.1 y = 8505.54x 0.99 Tyrosine 3.62 352.1 y = 2392.91x 0.97 Glutamine 2.44 317.1 y = 3891.44x 0.93 13 Arginine- C6 2.33 351.2 y = 476.85x 0.98 Lysine-D4 3.81 321.1 y = 1637.19x 0.98 Proline 2.98 286.1 y = 10992.47x 0.98 Aspartic acid 2.57 304.1 y = 4061.32x 0.92 Glutamic acid 2.65 318.1 y = 3228.65x 0.97 Histidine 1.91 326.1 y = 220.13x 0.98 Methionine 3.89 320.1 y = 5644.93x 0.96 Asparagine 2.29 303.1 y = 3236.46x 0.99 Leucine 6.18 302.1 y = 13907.29x 0.98 Tryptophan 6.57 375.1 –

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Supplementary Table 4 — Levels of free amino acids in iBAT of mice fed with SFA-CAA or SFA-EAA diet (iBAT aminograms)

SFA-CAA SFA-EAA

pmol/mg iBAT % pmol/mg iBAT % Fold change P Leucine 15.11  0.65 1.3 29.77  1.12 2.5 1.97 0.03 Lysine 68.61  1.06 6.0 23.84  1.51 1.9 0.35 0.04 Isoleucine 15.97  0.92 1.4 34.90  2.06 3.0 2.19 0.04 Valine 10.85  1.73 0.9 31.18  0.78 2.6 2.87 0.03 Threonine 21.84  1.81 1.9 157.88  2.81 13.0 7.23 0.02 Cysteine/cystine ------Histidine ------Phenylalanine 23.99  1.78 2.1 22.25  1.23 1.8 0.93 0.95 Methionine ------Tyrosine 31.10  3.72 2.7 29.22  2.20 2.4 0.94 0.98 Tryptophan ------Alanine 285.14  36.52 24.9 260.51  52.36 21.4 0.91 0.97 Arginine 37.56  1.42 3.3 70.73  2.12 5.8 1.88 0.04 Aspartic acid 20.73  2.34 1.8 19.22  3.73 1.6 0.93 0.62 Glutamic acid 371.08  25.40 32.4 343.99  26.84 28.3 0.93 0.92 Glycine 124.19  18.95 10.8 91.57  13.56 7.5 0.74 0.04 Proline 4.85  1.75 0.4 87.79  1.43 7.2 18.12 0.03 Serine 114.27  9.64 10.0 13.13  1.22 1.1 0.11 0.03

Amino acid levels in iBAT were measured with UPLC-MS/MS (in technical triplicates) after night consumption of SFA-CAA or SFA-EAA diets (6 weeks of treatment) (n = 5 mice per group). Increased amino acids in SFA-EAA- vs SFA-CAA-fed mice (in red); decreased amino acids in SFA- EAA- vs SFA-CAA-fed mice (in blue); undetected amino acid (in grey). The amino acid percentages (w/V) (grey shadow) were used to prepare EAA and CAA mixtures for the in vitro experiments. Values are expressed as mean ± SEM.

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Supplementary Table 5 — Primers for qRT-PCR

PCR Gene Primer Sense (5'-3') Primer Antisense (5'-3') Product Ta (°C) (bp) Acadl TGGGGACTTGCTCTCAACA GGCCTGTGCAATTGGAGTA 62 58 Atgl CAGCACATTTATCCCGGTGTAC AAATGCCGCCATCCACATAG 76 61 ATP2A1 TGCTGCTTTGCTCGTGTGGA CGGCCTTCTTCAGAGCAGGG 124 63 Cact GGTGGCTGTCCAGACAAACT TCCGTTTAAGAACCTCCTGG 122 59 CD137 CGTGCAGAACTCCTGTGATAAC GTCCACCTATGCTGGAGAAGG 103 60 CKMT2 GCATGGTGGCTGGTGATGAG AAACTGCCCGTGAGTAATCTTG 146 61 COX IV TGGGACTATGACAAGAATGAGTGG TTAGCATGGACCATTGGATACGG 113 63 CPT1a CGTTACTTCAAGGTCTGGCTCTAC GCTGCGGCTCTGAGGTGTC 103 62 CPT1b TGCCAGCCACAATTCACC GTCACTCACATAGTTAGTTGCC 167 58 CrAT GTTTCACCTGGGCCGCACTG TCAGTGTAGGCTCGGTGGGC 146 65 Cyt c ATAGGGGCATGTCACCTCAAAC GTGGTTAGCCATGACCTGAAAG 172 61 DIO2 GTCCGCAAATGACCCCTTT CCCACCCACTCTCTGACTTTC 65 59 eNOS CCTTCCGCTACCAGCCAGA CAGAGATCTTCACTGCATTGGCTA 105 61 FGF21 CTGCTGCTGGCTGTCTTCC GTCTTGGTCGTCATCTGTGTAG 124 60 GAMT GCACCTACCCTGCCTGACGG CCCCGGTCTTCAGCAAGCG 130 64 GAPDH AACTTTGGCATTGTGGAAGG ACACATTGGGGGTAGGAACA 183 60 GATm GACCTGGTCTTGTGCTCTCC GGGATGACTGGTGTTGGAGG 103 60 gDNA CGTGATCGTAGCGTCTGGTT GGTCGCGGTGTGGGCATTTG 108 62 Hsl GCTGGAGGAGTGTTTTTTTGC AGTTGAACCAAGCAGGTCACA 63 60 mGPDH TGCCCGGATACTACAGCCCT GCGTATCAGTGGTGCCAGCA 117 63 mtDNA ACATGCAAACCTCCATAGACCGG TCACTGCTGAGTCCCGTGGG 131 63 NRF-1 ACAGATAGTCCTGTCTGGGGAAA TGGTACATGCTCACAGGGATCT 99 61 Octn2 AAGACCTGCAGGAAGCTGAA TCCTTGTTTTTCGTGGGTGT 127 57 PGC-1α ACTATGAATCAAGCCACTACAGAC TTCATCCCTCTTGAGCCTTTCG 148 61 Pm20d1 GGCTTCATCTATGGCCGGGG ACACCTCCTCATCATGGCCCA 139 63 PRDM16 CTACAGGGTAGAAAAGCGGAAGG TGGTTTCTATGGCTGACATCTGG 110 63 Tbp CCCACAACTCTTCCATTCTCAAAC ACAGCCAAGATTCACGGTAGATAC 174 65 Tbx1 GGCAGGCAGACGAATGTTC TTGTCATCTACGGGCACAAAG 101 60 Tfam AAGACCTCGTTCAGCATATAACATT TTTTCCAAGCCTCATTTACAAGC 104 60 TFIIb TGGAGATTTGTCCACCATGA GAATTGCCAAACTCATCAAAACT 66 56 Tmem26 ACCCTGTCATCCCACAGA TGTTTGGTGGAGTCCTAA 119 55 UCP1 ACAGTACCCAAGCGTACCAAGC TGTTCAAAGCACACAAACATGATG 135 63

Ta, temperature of annealing; Acadl, acyl-coenzyme A dehydrogenase, long-chain; Atgl, adipose triglyceride lipase; ATP2A1, ATPase, Ca++ transporting, cardiac muscle, fast twitch 1; Cact, mitochondrial carnitine/acylcarnitine translocase; CD137, tumor necrosis factor receptor superfamily, member 9; CKMT2, , mitochondrial 2; COX IV, cytochrome c oxidase subunit IV; CPT1a, carnitine palmitoyltransferase 1a; CPT1b, carnitine palmitoyltransferase 1b; CrAT, carnitine acetyltransferase; Cyt c, cytochrome c; DIO2, type 2 iodothyronine deiodinase; eNOS, endothelial nitric oxide synthase; FGF21, fibroblast growth factor 21; GAMT, guanidinoacetate methyltransferase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GATm, glycine amidinotransferase; gDNA, genomic DNA ( 5 Actb); mGPDH, glycerol phosphate dehydrogenase 2; mtDNA, mitochondrial DNA; NRF1, nuclear respiratory factor 1; Octn2, organic cation transporter 2; PGC-1, peroxisome proliferator-activated receptor  coactivator 1; Pm20d1, peptidase M20 domain containing 1; PRDM16, PR domain containing 16; Tbp, TATA box binding protein; Tbx1, T-box 1; Tfam, transcription factor A; TFIIb, general transcription factor IIB; Tmem26, transmembrane protein 26; UCP1, uncoupling protein 1. 20 Diabetes Page 70 of 75

Supplementary Table 6 — Plasma analysis in mice fed with SFA-CAA and SFA-EAA diet for 6 weeks at room temperature

SFA-CAA SFA-EAA U mean SEM mean SEM P Alanine amino transferase (ALT) IU/L 46.5 6.0 27.0 6.5 0.16 Aspartate amino transferase IU/L 104.0 25.0 98.0 26.0 0.88 (AST) γ-Glutamyl transferase (GGT) IU/L 3.0 0.4 3.2 0.3 0.62 Alkaline phosphatase (ALK) IU/L 5.0 0.1 5.1 0.5 0.23 Creatin kinase (CK) IU/L 1713.5 716.5 644.5 112.5 0.28 Glucose mg/dL 151.5 9.5 159.0 36.0 0.86 P mg/dL 6.7 0.4 4.6 0.3 0.06 Ca mg/dL 4.0 0.2 4.3 0.1 0.08 Total bilirubin mg/dL 0.3 0.1 0.2 0.1 0.49 Total protein g/dL 5.0 0.5 5.2 0.2 0.33 Albumin g/dL 3.1 0.1 3.2 0.1 0.70 Urea nitrogen (BUN) mg/dL 18.5 1.5 15.5 0.5 0.20 Creatinine mg/dL 0.2 0.1 0.2 0.1 0.10 Na mEq/L 211.0 1.0 218.0 2.0 0.50 K mEq/L 6.4 0.6 5.6 0.4 0.38 Na/K ratio - 33.3 3.0 39.1 2.8 0.29 Cl mEq/L 79.0 1.0 81.0 1.0 0.29 Amylase IU/L 333.5 3.5 327.5 16.5 0.76 Total bile acids µmol/L 8.2 2.4 12.0 1.1 0.29 Insulin ng/mL 0.5 0.01 0.48 0.06 0.10 TSH pg/mL 352.5 50.3 376.2 48.6 0.12 Thyroxine (T4) ng/mL 57.2 1.8 47.8 0.8 0.06

Blood samples were collected by submandibular venipuncture, during the same time interval after a fasting period of 5 h. Clinical chemistry parameters and hormone levels in mice fed with SFA-CAA or SFA-EAA for 6 weeks at room temperature. Values are expressed as the mean ± SEM. P values are show as differences between dietary regimens (n = 4 mice per group).

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Supplementary Table 7 — Spearman’s correlation analysis

BW Adiposity GTT Liver weight Liver OCR Spearman P Spearman P Spearman P Spearman P Spearman P Alanine -0.11 0.70 -0.16 0.56 0.07 0.81 -0.32 0.25 0.35 0.20 Arginine 0.47 0.08 0.57 0.03 -0.26 0.35 0.45 0.089 -0.51 0.054 Aspartate 0.03 0.92 -0.05 0.86 -0.13 0.64 -0.20 0.48 0.24 0.39 Asparagine 0.08 0.79 0.20 0.47 0.08 0.77 -0.16 0.58 0.24 0.38 Glutamate -0.70 0.004 -0.54 0.04 -0.02 0.95 -0.74 0.002 0.58 0.02 Glutamine -0.02 0.940 0.13 0.64 0.19 0.50 -0.14 0.62 0.30 0.27 Glycine 0.22 0.44 0.27 0.33 0.06 0.84 -0.18 0.53 0.06 0.82 Histidine -0.77 0.001 -0.64 0.01 0.08 0.79 -0.85 0.0001 0.61 0.02 Isoleucine -0.55 0.03 -0.44 0.101 0.34 0.22 -0.19 0.51 0.24 0.40 Leucine -0.47 0.08 -0.52 0.05 0.12 0.67 -0.17 0.55 0.19 0.49 Lysine -0.29 0.30 -0.06 0.83 0.05 0.86 -0.47 0.08 0.41 0.13 Methionine 0.18 0.52 0.32 0.24 -0.39 0.15 0.13 0.65 -0.26 0.35 Phenylalanine -0.07 0.80 0.04 0.88 -0.52 0.16 -0.08 0.77 -0.11 0.70 Proline 0.17 0.54 -0.01 0.96 -0.33 0.24 0.05 0.86 -0.10 0.71 Serine 0.14 0.63 0.13 0.64 0.13 0.65 -0.27 0.33 0.08 0.77 Threonine -0.67 0.006 -0.69 0.0048 0.07 0.81 -0.40 0.14 0.45 0.09 Tyrosine 0.49 0.06 0.68 0.005 -0.22 0.43 0.19 0.50 -0.45 0.10 Valine -0.61 0.016 -0.63 0.0125 -0.04 0.88 -0.30 0.28 0.18 0.53

Amino acid plasma levels were correlated with body weight (BW), adiposity [i.e., epididymal white adipose tissue (eWAT) weight], glucose homeostasis [i.e., glucose tolerance test (GTT)], and liver parameters [liver weight and mitochondrial oxygen consumption rates (OCR)] measured after consumption of diets for six weeks. Positively and negatively Spearman correlation coefficient (r) with P values are shown.

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Supplementary Table 8 — Influence of diets on energy metabolism

2 weeks 6 weeks SFA-CAA SFA-EAA SFA-CAA SFA-EAA

n = 7 n = 7 n = 6 n = 6 P P

VO2 (mL/h) 99.54 2.75 94.59 1.38 0.13 75.83 1.55 87.75 1.77 0.001

VO2 BW (mL/h/kg) 3691.33 128.13 3714.70 40.13 0.87 3341.69 36.69 3677.50 49.52 0.001

VO2 LM (mL/h/kg) 4946.10 125.13 4697.21 77.21 0.12 4307.04 56.77 4652.97 81.41 0.009 EE (kcal/h) 0.44 0.03 0.45 0.01 0.51 0.38 0.01 0.44 0.01 0.001

EEBW (kcal/h/kg) 18.21 0.64 18.18 0.20 0.97 17.20 0.29 18.08 0.22 0.042

EELM (kcal/h/kg) 24.40 0.62 22.99 0.40 0.08 22.87 0.40 25.45 0.59 0.007 Movement (counts/h) 14368.0 1131.9 15243.8 1549.1 0.65 21291.4 1369.9 19331.0 1148.7 0.334 HFD-CAA HFD-EAA HFD-CAA HFD-EAA

n = 5 n = 6 n = 5 n = 6 P P

VO2 (mL/h) 115.10 5.92 108.07 3.56 0.32 104.41 3.29 117.80 4.10 0.04

VO2 BW (mL/h/kg) 2463.27 76.77 2548.40 125.01 0.58 2281.02 60.83 2569.29 95.39 0.04

VO2 LM (mL/h/kg) 4836.70 149.38 4655.35 111.35 0.36 4401.70 87.59 5060.49 201.82 0.02 EE (kcal/h) 0.57 0.03 0.51 0.02 0.18 0.50 0.02 0.58 0.02 0.02

EEBW (kcal/h/kg) 11.89 0.37 12.16 0.60 0.71 11.76 0.42 13.56 0.53 0.03

EELM (kcal/h/kg) 23.34 0.74 22.21 0.53 0.25 20.92 0.48 23.02 0.52 0.02 Movement (counts/h) 12648.6 1766.2 14106.7 1362.8 0.55 16573.9 3628.7 15348.0 3088.3 0.80

Average 24 h oxygen consumption (VO2) and energy expenditure (EE) of mice fed for 2 or 6 weeks with SFA-CAA, SFA-EAA, HFD-CAA, or HFD-EAA. VO2 is expressed as mL/h/mouse, while EE is expressed as EE kcal/mouse/h. VO2 BW, VO2 LM, EEBW, and EELM are normalized by body weight (BW) and lean mass (LM), respectively. Average 24 h total movement is reported as activity number of x and y-axis infrared photobeam breaks (counts) per h. Values are reported as mean ± SEM. A separate experiment of indirect calorimetry was performed for each diet type (SFA or HFD) and time interval (2 or 6 weeks); thus P values are shown as differences between the single diet at the same time interval.

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Supplementary Table 9 — Relative abundance of microbial taxa at the genus level in gut microbiota of mice fed with SFA, SFA-CAA, and SFA-EAA diets for 6 weeks

SFA SFA-CAA SFA-EAA SFA vs SFA-CAA SFA vs SFA-EAA SFA-EAA vs SFA-CAA Taxa (Genus) mean SD mean SD mean SD P FDR P P FDR P P FDR P Acinetobacter 0.00160 0.00237 0.00605 0.00712 0.00122 0.00176 0.1545 0.6018 0.8398 1.0000 0.2257 0.4657 Actinomyces 0.00071 0.00189 0.00022 0.00065 0.00000 0.00000 0.8072 0.9213 0.2636 0.5979 0.3173 0.4657 Adlercreutzia 0.17391 0.12530 0.17204 0.07809 0.07314 0.04231 0.7898 0.9213 0.3284 0.6408 0.0963 0.3627 AF12 0.00200 0.00530 0.00000 0.00000 0.00000 0.00000 0.2636 0.6018 0.2636 0.5979 0.9422 1.0000 Akkermansia 14.8044 12.7593 22.3752 12.8806 29.2526 14.5437 0.2481 0.6018 0.0129 0.2420 0.0413 0.2913 Allobaculum 3.37911 4.59730 2.75129 3.48290 13.48335 5.82501 0.9292 0.9969 0.0005 0.0299 0.0003 0.0322 Anaerobacillus 0.00302 0.00660 0.00021 0.00063 0.00040 0.00120 0.3384 0.6129 0.4119 0.6808 0.9422 1.0000 Anaerofustis 0.00391 0.00544 0.00101 0.00162 0.00000 0.00000 0.4273 0.6615 0.0402 0.3788 0.0682 0.3279 Anaeroplasma 0.00000 0.00000 0.00000 0.00000 0.38383 1.15148 0.9352 1.0000 0.3711 0.6408 0.3173 0.4657 Anaerotruncus 0.03695 0.02349 0.06746 0.04771 0.02135 0.00710 0.1974 0.6018 0.8590 1.0000 0.0821 0.3436 Bacillus 0.00098 0.00170 0.00461 0.00975 0.00168 0.00504 0.5740 0.7814 0.4941 0.7567 0.3043 0.4657 Bacteroides 22.5826 9.78316 15.1754 12.7729 9.58613 7.19427 0.1309 0.6018 0.1551 0.5979 0.4057 0.5659 Bifidobacterium 0.00048 0.00128 0.00065 0.00141 3.55909 5.40134 0.6816 0.8710 0.0033 0.0919 0.0021 0.0705 Bilophila 0.03123 0.02346 0.04572 0.03626 0.02270 0.02689 0.3743 0.6129 0.3711 1.0000 0.2899 0.4657 Blautia 0.00026 0.00069 0.00045 0.00135 0.00037 0.00112 0.9352 0.9969 0.9352 1.0000 0.9422 1.0000 Brachybacterium 0.00084 0.00222 0.00000 0.00000 0.00000 0.00000 0.2636 0.6018 0.2636 0.5979 0.9422 1.0000 Brevundimonas 0.00227 0.00410 0.00000 0.00000 0.00033 0.00099 0.1037 0.6018 0.3384 0.6408 0.3173 0.4657 Candidatus arthromitus 0.08854 0.23426 0.00000 0.00000 0.00261 0.00782 0.2636 0.6018 0.8072 0.9915 0.3173 0.4657 Chryseobacterium 0.00212 0.00231 0.00191 0.00451 0.00195 0.00226 0.3408 0.6129 0.7758 0.9915 0.2143 0.4657 Citrobacter 0.00000 0.00000 0.00133 0.00205 0.00000 0.00000 0.1008 0.6018 0.3711 1.0000 0.0682 0.3279 Coprobacillus 0.00026 0.00069 0.00986 0.01145 0.01212 0.02616 0.0262 0.4632 0.1772 0.5979 0.4673 0.6362 Coprococcus 0.99149 0.48498 0.89902 0.51838 1.39069 1.62534 0.3743 0.6129 0.2863 0.6105 0.0821 0.3436 Corynebacterium 0.00084 0.00222 0.00000 0.00000 0.00000 0.00000 0.2636 0.6018 0.2636 0.5979 0.9422 1.0000 Curvibacter 0.00000 0.00000 0.00000 0.00000 0.00037 0.00112 0.9352 1.0000 0.3711 0.6408 0.3173 0.4657 Dehalobacterium 0.74957 0.39910 0.93851 0.51006 0.36417 0.26029 0.3284 0.6129 0.1551 0.5979 0.0696 0.3279 Delftia 0.00198 0.00212 0.00510 0.00374 0.00171 0.00157 0.0237 0.4632 0.2646 0.5979 0.1455 0.3797 Desulfovermiculus 0.00073 0.00193 0.00021 0.00063 0.00000 0.00000 0.8072 0.9213 0.2636 0.5979 0.3173 0.4657 Desulfovibrio 0.06065 0.16047 0.06638 0.13276 0.00000 0.00000 0.7844 0.9213 0.2636 0.5979 0.1468 0.3797 Dorea 0.11502 0.04637 0.22963 0.29937 0.07852 0.10686 0.2863 0.6105 0.1097 0.5392 0.0588 0.3279 Diabetes Page 74 of 75

Enterobacter 0.00088 0.00233 0.00083 0.00166 0.00000 0.00000 0.7844 0.9213 0.2636 0.5979 0.1468 0.3797 Enterococcus 0.02707 0.02873 0.07036 0.07258 0.01009 0.00541 0.0263 0.4632 0.7223 0.9490 0.0025 0.0705 Exiguobacterium 0.00000 0.00000 0.00135 0.00405 0.00000 0.00000 0.3711 0.6129 0.3711 1.0000 0.3173 0.4657 Facklamia 0.00000 0.00000 0.00086 0.00173 0.00000 0.00000 0.1931 0.6018 0.3711 1.0000 0.1468 0.3797 Flexispira 0.00052 0.00138 0.00000 0.00000 0.00000 0.00000 0.2636 0.6018 0.2636 0.5979 0.9422 1.0000 Helicobacter 2.03057 3.76863 0.05608 0.16305 0.64017 1.55791 0.4915 0.7006 0.7328 0.9518 0.2494 0.4657 Kocuria 0.00212 0.00240 0.00445 0.00515 0.00134 0.00236 0.3140 0.6129 0.6860 0.9252 0.1826 0.4128 Lactobacillus 0.72554 1.34432 0.69220 0.53928 4.34119 5.90851 0.0410 0.4632 0.0045 0.1009 0.0082 0.1316 Lactococcus 4.00347 6.68594 0.18356 0.10794 0.17767 0.37243 0.0004 0.0429 0.0019 0.0705 0.0963 0.3627 Leuconostoc 0.00044 0.00116 0.00175 0.00216 0.00069 0.00119 0.1772 0.6018 0.4278 0.6808 0.5946 0.7576 Microbacterium 0.00000 0.00000 0.00000 0.00000 0.00060 0.00121 0.9352 1.0000 0.1931 0.5979 0.1468 0.3797 Mitsuokella 0.00084 0.00222 0.00000 0.00000 0.00000 0.00000 0.2636 0.6018 0.2636 0.5979 0.9422 1.0000 Mucispirillum 0.16651 0.28652 1.17904 1.33989 0.83264 1.28583 0.1662 0.6018 0.0907 0.4917 0.9397 1.0000 Ochrobactrum 0.00210 0.00555 0.00043 0.00086 0.00037 0.00112 0.7844 0.9213 0.8072 0.9915 0.6267 0.7782 Odoribacter 1.77718 2.90161 6.42061 5.90189 1.58773 1.40149 0.0209 0.4632 0.2481 0.5979 0.0963 0.3627 Oscillospira 10.68389 5.36761 9.71479 4.11254 4.71619 3.33414 0.4772 0.6913 0.1826 0.5979 0.0821 0.3436 Parabacteroides 22.55795 16.67867 17.79957 9.94875 12.68725 6.17695 0.7898 0.9213 0.8590 1.0000 0.6501 0.7986 Paracoccus 0.00000 0.00000 0.00041 0.00124 0.00026 0.00079 0.3711 0.6129 0.3711 0.6408 0.9422 1.0000 Paraprevotella 0.00000 0.00000 0.07330 0.15711 0.00000 0.00000 0.1931 0.6018 0.0907 1.0000 0.1468 0.3797 Prevotella 0.00000 0.00000 0.00000 0.00000 0.00470 0.00986 0.9352 1.0000 0.1931 0.5979 0.1468 0.3797 Propionibacterium 0.00000 0.00000 0.00112 0.00270 0.00000 0.00000 0.1931 0.6018 0.0907 1.0000 0.1468 0.3797 Pseudomonas 0.00084 0.00222 0.00157 0.00340 0.00205 0.00231 0.6816 0.8710 0.1249 0.5880 0.1558 0.3828 Pseudoxanthomonas 0.00000 0.00000 0.00071 0.00153 0.00000 0.00000 0.1931 0.6018 0.0907 1.0000 0.1468 0.3797 Rhodococcus 0.00000 0.00000 0.00078 0.00175 0.00034 0.00101 0.1931 0.6018 0.3711 0.6408 0.6267 0.7782 Roseburia 0.00000 0.00000 0.07609 0.18854 0.00019 0.00056 0.1931 0.6018 0.3711 0.6408 0.4656 0.6362 Rothia 0.00084 0.00222 0.00000 0.00000 0.00000 0.00000 0.2636 0.6018 0.2636 0.5979 0.9422 1.0000 Ruminococcus 3.52180 2.12668 4.36248 2.04570 2.51037 2.05136 0.4772 0.6913 0.7898 0.9915 0.5453 0.7166 Salana 0.00071 0.00189 0.00000 0.00000 0.00026 0.00079 0.2636 0.6018 0.9352 1.0000 0.3173 0.4657 Serratia 0.00674 0.00730 0.01162 0.00746 0.00393 0.00279 0.1301 0.6018 0.5901 0.8286 0.1735 0.4000 SMB53 0.00942 0.01527 0.00168 0.00361 0.00267 0.00414 0.0689 0.6018 0.5690 0.8139 0.2257 0.4657 Sphingobacterium 0.00000 0.00000 0.00100 0.00299 0.00000 0.00000 0.3711 0.6129 0.0907 1.0000 0.3173 0.4657 Staphylococcus 0.01643 0.01300 0.02407 0.01899 0.00347 0.00406 0.4499 0.6870 0.0725 0.4494 0.0182 0.1884 Streptococcus 0.02200 0.03673 0.01224 0.00764 0.00430 0.00477 0.3733 0.6129 0.5264 0.7736 0.1478 0.3797 Sutterella 0.81837 1.05663 3.53592 7.35599 6.87912 6.33044 0.2481 0.6018 0.0005 0.0299 0.0041 0.0920 Turicibacter 1.69882 3.94318 0.29104 0.58247 1.44283 2.06308 0.0854 0.6018 0.6877 0.9252 0.0692 0.3279 Other 4.50579 3.64642 5.96632 3.69171 3.50317 4.03575 0.9352 0.9969 0.1059 0.9915 0.9422 0.1884

25 Page 75 of 75 Diabetes

[Prevotella] 0.51313 1.16283 0.89861 0.25728 0.68826 0.98051 0.0179 0.4632 0.2431 0.5979 0.1730 0.4000 [Ruminococcus] 2.46031 0.66873 2.98674 3.69159 1.07346 0.92375 0.9292 0.9969 0.0330 0.3387 0.0233 0.1884 Unassigned 0.12355 0.31389 2.87607 2.19429 0.20738 0.25575 0.1309 0.6018 0.1059 0.9915 0.1124 0.3797

Data are presented as percentage (mean ± SD). The non-parametric Kruskal Wallis test was used for the comparison of relative abundances of microbial taxa between groups, and the resulting P values were corrected (Bonferroni correction) for multiple testing controlling the false discovery rate (FDR P value).

26 Diabetes Page 76 of 75

Supplementary Table 10 — Amino acid amounts in SFA-EAA diet compared to the amino acid requirements of mice

SFA-EAA (RD number: D14032501) NRC Amino Grams Guidelines Ingredients g/kg diet acids in formulaa (g/kg diet)b Protein Leucine 59.77 7.0 Casein 200 Valine 29.91 5.0 Lysine 31.32 4.0 Carbohydrates Isoleucine 29.78 4.0 Corn Starch 452 Threonine 17.01 4.0 Maltodextrin 75 Histidine 7.46 2.0 Sucrose 173 Cysteine/cystine 7.21 Cellulose 50 Phenylalanine 5.3 7.6 Methionine 2.82 5.0 Fat Tyrosine 1.99 Soybean oil 25 Tryptophan 1.11 1.0 Lard 20 Alanine 0.4 Mineral Mix S10026 10 Arginine 0.44

CaHPO4 13 Aspartic acid 0.9

CaCO3 5.5 Glutamic acid 2.82 Potassium Citrate 16.5 Glycine 0.22 Vitamin Mix 10 Proline 1.31 Choline Bitartrate 2 Serine 0.74 aTotal amino acid amount derived from casein (13 g) and EAAm; bessential amino acid amounts for mice as recommended by the U.S. National Research Council (NRC) (Nutrient Requirements of Laboratory Animals. Fourth Revised Edition, 1995).