High-protein diet prevents fat mass increase after dieting by counteracting Lactobacillus-enhanced lipid absorption

Refeeding after dieting induces fat mass increase

To study the effect of refeeding after dieting on fat mass, we fed mice with 1-d food in 3 d or 4-d food in 6 d as models of SDR and then fed ad libitum (Fig. 1a). Typically, feeding with 10%, 25% and 65% food from day 1 to day 3, respectively led to a quick decrease in fat mass as expected (Fig. 1b). Notably, refeeding ad libitum after feeding with 10–25–65% food for 3 d led to quick fat mass accumulation (Fig. 1b and Extended Data Fig. 1a–d). Moreover, refeeding after feeding with 65–25–10% or 33.3% food daily for 3 d or 66.7% food daily for 6 d, also led to similar quick fat mass accumulation (Fig. 1c–e and Extended Data Fig. 1b–d). To further confirm the effect of refeeding after SDR on fat mass, we killed and dissected the mice. Refeeding after feeding with 10–25–65% food increased the size and weight of inguinal WAT (iWAT) and epididymal WAT (eWAT) (Fig. 1f,g and Extended Data Fig. 1f–j). Hematoxylin and eosin (H&E) staining revealed that refeeding after the SDR markedly increased the size of adipocytes in iWAT and eWAT (Fig. 1h and Extended Data Fig. 1k–m). Consistently, refeeding after the SDR led to the hypertrophy of interscapular brown adipose tissue (iBAT) and the accumulation of enlarged lipid droplets in iBAT cells (Extended Data Fig. 2a–c) and induced hepatic steatosis (Extended Data Fig. 2d,e).

Fig. 1: Refeeding after various types of dietary restriction induces quick fat mass accumulation. a, Experimental design of SDR in mice feeding with 1-d food in 3 d or 4-d food in 6 d. AL, ad libitum; SDR(33.3% × 3), 33.3% food daily provided from day 1 to 3; SDR(66.7% × 6), 66.7% food daily provided from day 1 to 6. b–e, Refeeding after feeding with 1-d food in 3 d (b–d) or 4-d food in 6 d (e) markedly increased body fat mass, n = 9 biologically independent mice for AL, SDR(10–25–65%) and SDR(66.7% × 6), n = 7 for SDR(65–25–10%), and n = 8 for SDR(33.3% × 3). f, Representative images of isolated iWAT from the SDR(10–25–65%) group. The size for each square is 0.5 cm × 0.5 cm. D4D, day 4 at dark phase. g, The weight of iWAT in f (n = 12 biologically independent mice per group). h, Representative images of H&E-stained sections of iWAT in f. Scale bar, 50 μm. i–k, Refeeding after feeding with 2-d food in 3 d (i,j) or 1-d food in 2 d (k) significantly increased body fat mass; n = 8 biologically independent mice for AL, n = 9 for other groups. l,m, Refeeding after feeding with 66.7% food daily for 12 d (l) or 24 d (m) markedly increased body fat mass (n = 9 biologically independent mice per group). n, Refeeding after alternate-day fasting for 15 cycles markedly increased body fat mass (n = 9 biologically independent mice per group). The data shown in b–e, i–k or l–n were performed simultaneously with a single control experiment. Data are presented as mean ± s.d. Statistical significance was determined by two-tailed Student’s t-test. a, P < 0.05; b, P < 0.01; c, ***P < 0.001; versus AL or D0 group. Exact P values can be found in Source Data Fig. 1. Source data Full size image

To sufficiently investigate the effect of refeeding after DR on fat mass, we fed mice with 2-d food in 3 d, 1-d food in 2 d or 66.7% food daily for 12 d or as long as 24 d and then fed ad libitum (Extended Data Fig. 3a,g). Similarly, refeeding after these types of DR all led to quick fat mass accumulation (Fig. 1i–m and Extended Data Fig. 3b–e,h–k). Alternate-day fasting as an intermittent fasting regime has been considered to be as beneficial for reducing fat mass as daily DR51. We found that refeeding after alternate-day fasting also led to similar quick fat mass accumulation as refeeding after other types of DR (Fig. 1n and Extended Data Fig. 3g,m–p). These results demonstrate that refeeding after dieting leads to quick fat mass accumulation in mice.

Enhanced intestinal lipid absorption contributes to fat mass increase after SDR

To reveal the mechanism of fat mass increase after SDR, first we analyzed the energy expenditure. As shown in Fig. 2a,b and Extended Data Fig. 3r–y, we found that energy expenditure, physical activity and body temperature were not significantly altered after SDR. These data demonstrate that quick fat mass accumulation after SDR is not due to a decrease in energy expenditure.

Fig. 2: Refeeding after SDR upregulates intestinal lipid absorption, increases fatty acid uptake and lipid synthesis in WAT and decreases total lipid oxidation. a,b, Average energy expenditure (EE) from day 4 to day 5 in mice fed ad libitum (AL) or mice in the SDR(10–25–65%) (a) or SDR(33.3% × 3) group (b), n = 7 and 6 biologically independent mice for AL in a and b, respectively, n = 8 for SDR(10–25–65%), n = 6 for SDR(33.3% × 3). c, Hypothalamic mRNA levels of orexigenic genes Agrp, Npy and anorexigenic genes Pomc, Cart of mice in the SDR(10–25–65%) group (n = 8 biologically independent samples per group). d, Cumulative food intake of mice fed AL (n = 9 biologically independent mice), in the SDR(10–25–65%) group (n = 9) or SDR(33.3% × 3) group (n = 8). e,f, Refeeding with food as much as in D0 after feeding with 10%, 25% and 65% food (e) or 33.3% food (f) daily from day 1 to day 3 markedly increased body fat mass (n = 9 biologically independent mice per group). SDR(10–25–65%)−100% × 23 and SDR(33.3% × 3)−100% × 23, 10%, 25% and 65% food or 33.3% food daily provided from day 1 to day 3 and subsequently 100% food as much as in D0 daily provided for 23 d. g, Cumulative food intake of mice in e,f (n = 9 biologically independent mice per group). h, Fecal TG content in SDR(10–25–65%) group (n = 9 biologically independent samples for D0, D1, D2, D3, D4, D6 and D9, n = 8 for D5, n = 10 for D7 and D8). i, Representative images of Oil-Red-O-stained sections of small intestinal villi at ZT10 (D0L–D9L) or ZT15 (D4D, day 4 at dark phase) in SDR(10–25–65%) group. j, TG content of small intestinal tissues in i (n = 7 biologically independent samples for D0, n = 6 for D1, D6 and D9, n = 8 for D2, D3, D4D and D4). k, Post-gavage serum TG levels in mice fed AL or in the SDR(10–25–65%) group. n = 7, 8, 7, 8 and 8 biologically independent samples for AL at 0, 1, 2, 4, 6 h, respectively, n = 8, 8, 7, 7, 8 for SDR(10–25–65%). l, Fecal BODIPY concentrations of mice fed AL or in the SDR(10–25–65%) group after oral gavage with olive oil containing BODIPY-labeled fatty acid for 10 min to 2 h at D5 (n = 9 biologically independent mice per group). m, Representative fluorescent section images of small intestinal villi from mice fed AL or in the SDR(10–25–65%) group after oral gavage as in l for 2 h at D5. n,o, Small intestine (n) and serum (o) BODIPY concentrations of mice in m (n = 9 biologically independent mice per group). p, Representative fluorescent section images of iWAT and eWAT from mice in m. q, BODIPY concentrations of iWAT and eWAT in p (n = 9 biologically independent mice per group). r,s, Respiratory exchange ratio (RER) of mice fed AL or mice in SDR(10–25–65%) (r) or SDR(33.3% × 3) (s) group. n = 7 and 6 biologically independent mice for AL in r and s, respectively, n = 8 for SDR(10–25–65%), n = 6 for SDR(33.3% × 3). t,u, Food intake of mice fed AL or mice in the SDR(10–25–65%) (t) or SDR(33.3% × 3) (u) group (n = 8 biologically independent mice per group). The data shown in e,f were performed simultaneously with a single control experiment. Data are presented as mean ± s.d. Statistical significance was determined by two-tailed Student’s t-test. a, *P < 0.05; b, **P < 0.01; c, ***P < 0.001; NS, not significant; versus AL or D0 group. Exact P values can be found in Source Data Fig. 2. Source data Full size image

Then we investigated whether food intake was involved in quick fat mass accumulation after SDR. As shown in Fig. 2c, refeeding after SDR significantly increased hypothalamic messenger RNA levels of orexigenic genes Agrp and Npy and decreased hypothalamic mRNA levels of anorexigenic genes Pomc and Cart. Consistently, we found that food intake during the first several days of refeeding after dieting is markedly increased (Extended Data Figs. 1e and 3f,l,q). To determine whether increased food intake contributes to the quick fat mass accumulation after SDR, we analyzed cumulative food intake. Notably, cumulative food intake of mice for all the tested types of dieting was similar to or even lower than that of mice fed ad libitum (Fig. 2d and Extended Data Fig. 4a–d), indicating that cumulative food intake has no significant positive contribution to quick fat mass accumulation after SDR. To investigate the effect of increased food intake during the first several days of refeeding after SDR on fat mass, after feeding with 10–25–65% food or 33.3% × 3 food for 3 d, we fed the mice with 100% food per day as consumed just before SDR (Extended Data Fig. 4e,f). Similarly, refeeding with 100% food after SDR also led to quick fat mass accumulation (Fig. 2e,f and Extended Data Fig. 4g–j), although the cumulative food intake of mice in these two SDR groups was significantly lower than that of the mice fed ad libitum (Fig. 2g). These data demonstrate that the increased food intake during the first several days of refeeding is not the major cause of quick fat mass accumulation after SDR.

To further investigate the mechanism of quick fat mass accumulation after SDR, we analyzed whether increased intestinal lipid absorption was involved. We found that fecal triglyceride (TG) of mice fed with 10–25–65% food for 3 d was significantly decreased (Fig. 2h). Moreover, both Oil-Red-O-stained sections of small intestinal villi and direct quantification of small intestinal TG show that small intestinal TG was markedly increased after SDR (Fig. 2i,j). Gavage of olive oil and measurement of serum TG levels also suggest enhanced intestinal lipid absorption after SDR (Fig. 2k). To clearly monitor the intestinal lipid absorption, gavage of BODIPY-labeled fatty acid combined with olive oil was performed. The fecal BODIPY levels dramatically decreased after SDR (Fig. 2l) and the fluorescence intensity of villus sections from proximal jejunum was significantly increased after SDR (Fig. 2m), which is further confirmed by direct quantification of intestinal and serum BODIPY levels (Fig. 2n,o). These data show that enhanced intestinal lipid absorption contributes to the quick fat mass accumulation after SDR.

To further understand how increased intestinal lipid absorption finally leads to quick fat mass accumulation after SDR, we analyzed the fatty acid uptake and lipid synthesis in WAT. Gavage of BODIPY-labeled fatty acid showed that the fluorescence intensity of iWAT and eWAT sections significantly increased after SDR (Fig. 2p), which is further confirmed by direct quantification of BODIPY levels (Fig. 2q). The results from gavage of BODIPY-labeled fatty acid demonstrate that refeeding after SDR increases fatty acid uptake in WAT. Furthermore, fatty acid and TG synthesis significantly increased in iWAT after SDR when analyzed by high-throughput RNA sequencing (Extended Data Fig. 4k). Moreover, an increase in the respiratory exchange ratio after SDR was observed (Fig. 2r,s and Extended Data Fig. 4l,m), indicating a decreased total lipid oxidation in mice after SDR52. In addition, food intake in both dark and light phase significantly increased after SDR (Fig. 2t,u and Extended Data Fig. 4n,o). These data demonstrate that increased fatty acid uptake and lipid synthesis in WAT and decreased total lipid oxidation contribute to the quick fat mass accumulation after SDR.

Refeeding with high-protein diet blocks fat mass increase after dieting

To seek potential dietary interventions to prevent fat mass increase after dieting, we measured serum metabolites by non-targeted metabolomics before, during and after SDR. Differential serum metabolites were majorly concentrated on protein digestion and absorption, amino acid metabolism, biosynthesis of amino acids and related pathways (Fig. 3a,b) and the levels of most amino acids were obviously changed (Extended Data Fig. 5a). Further quantification of serum amino acids by liquid chromatography−tandem mass spectrometry (LC–MS/MS) showed that essential amino acids (EAAs) except methionine and some semi-essential and non-essential amino acids were significantly increased during and after SDR (Fig. 3c and Extended Data Fig. 5b). To further explore the potential effect of amino acids on quick fat mass accumulation after SDR, we fed mice with an HP diet, low-protein diet or normal-protein (NP) diet supplemented with EAAs after feeding with 10–25–65% food for 3 d (Fig. 3d). As shown in Fig. 3e and Extended Data Fig. 5c–f, all three diets fed after SDR prevented quick fat mass accumulation and especially the HP diet even partially maintained the fat loss induced by SDR. The effect of feeding with the HP diet after SDR on fat mass was further confirmed by the size and weight of iWAT and eWAT (Fig. 3f,g and Extended Data Fig. 5g–i). H&E staining also revealed that feeding with an HP diet after SDR markedly blocked the increase in adipocyte size (Fig. 3h and Extended Data Fig. 5j–l). Consistently, refeeding with an HP diet after the SDR markedly blocked the hypertrophy of iBAT and the accumulation of enlarged lipid droplets in iBAT cells (Extended Data Fig. 5m–o) and inhibited hepatic steatosis (Extended Data Fig. 5p,q).

Fig. 3: Refeeding with high-protein diet efficiently maintains fat mass loss induced by dieting. a,b, KEGG pathway enrichment analysis based on the significantly altered metabolites in serum of mice in SDR(10–25–65%) group (n = 4 biologically independent mice per group). c, Serum essential amino acid levels of mice in SDR(10–25–65%) group (n = 4 biologically independent mice for D0 and D4, n = 3 for D2). d, Experimental design of mice refeeding with different diets after feeding with 10%, 25% and 65% NP diet provided from day 1 to day 3. LP, low-protein diet. e, An HP diet maintained fat mass loss induced by SDR with 10%, 25% and 65% food intake from day 1 to day 3. n = 18 biologically independent mice for AL-NP and SDR-NP, n = 9 for other groups. f, Representative images of isolated iWAT from mice as treated with NP or HP diet after SDR in e. g, The weight of iWAT from mice in f (n = 10 biologically independent mice for D0, n = 6 for other groups). h, Representative images of H&E-stained sections of iWAT in f. i,j, HP diet maintained fat mass loss induced by SDR with 33.3% food intake daily for 3 d (i) or DR with 66.7% food intake daily for 12 d (j). n = 9 and 8 biologically independent mice for AL-NP in i and j, respectively, n = 9 for other groups. k, HP diet maintained fat mass loss in female mice induced by SDR with 10%, 25% and 65% food intake from day 1 to day 3 (n = 10 biologically independent mice per group). Data are presented as mean ± s.d. Statistical significance was determined by two-tailed Student’s t-test. a, *P < 0.05; b, **P < 0.01; c, ***P < 0.001; except indicated, SDR-HP group versus AL-NP or SDR-NP group, DR-HP group versus AL-NP or DR-NP group. Exact P values can be found in Source Data Fig. 3. Source data Full size image

To further confirm the reverse effect of HP diet on fat gain after dieting, we fed mice with HP diet after feeding with 33.3% food daily for 3 d or 66.7% food daily for 12 d. As expected, feeding with HP diet after these two types of dieting prevented quick fat mass accumulation after dieting and even partially maintained the fat-loss effect of dieting (Fig. 3i,j and Extended Data Fig. 6a–h). In addition, feeding with HP diet after feeding with 10%–25%–65% food for 3 d in female mice also had a similar effect (Fig. 3k and Extended Data Fig. 6i–l).

These data show that fat mass increase after dieting can be prevented by dietary intervention, especially in the form of an HP diet.

High-protein diet after SDR attenuates intestinal lipid absorption

To investigate the mechanism of how HP diets prevent quick fat mass accumulation after SDR, we analyzed the energy expenditure. As shown in Fig. 4a, energy expenditure in mice fed HP diet after SDR was significantly lower than that of mice fed NP diet after SDR. These results show that the inhibitory effect of the HP diet on quick fat mass accumulation after SDR was not due to the increase in energy expenditure.

Fig. 4: Refeeding with high-protein diet after SDR downregulates intestinal lipid absorption, decreases fatty acid uptake and lipid synthesis in WAT and increases total lipid oxidation. a, Energy expenditure (EE) of mice fed ad libitum (AL) (n = 4 biologically independent mice) or fed with NP (n = 6) or HP (n = 6) diet after SDR with 10%, 25% and 65% food from day 1 to day 3. b, Cumulative food intake of mice as treated in a (AL-NP, n = 18 biologically independent mice; SDR-NP, n = 18; SDR-HP, n = 9). c, Experimental design of pair-feeding studies. Mice were pair-fed to the amount of daily caloric intake consumed by those in SDR-HP group. HF, high-fat diet; PF, pair-feeding. d, Daily caloric intake of mice in different regimens in c. n = 11 biologically independent mice for AL-NP, SDR-NP and SDR-NP(PF), n = 10 for SDR-HP and SDR-HF(PF). e, Pair-feeding with NP diet or high-fat diet after SDR markedly increased body fat mass. n = 11 mice for AL-NP, SDR-NP and SDR-NP(PF), n = 10 for SDR-HP and SDR-HF(PF). f, Fecal TG of mice as treated in a (n = 10, 7, 10, 9, 9, 9 and 8 biologically independent samples for SDR-NP on D0, D4, D5, D6, D7, D8 and D9, respectively, n = 10, 7, 9, 8, 9, 10 and 10 for SDR-HP). g,h, LCFA composition of cecal feces from mice as treated in a (n = 10 biologically independent mice per group). i,j, Total intestinal (i) and serum (j) bile acid concentrations of mice as treated in a. n = 8 and 10 biologically independent samples for AL-NP(D0) in i and j, respectively, n = 4 and 5 for SDR-NP(D4) or SDR-HP(D4) in i and j, respectively. k–n, Intestinal and serum primary (k,l) and secondary (m,n) bile acid composition from mice in (i,j). n = 8 and 10 biologically independent samples for AL-NP(D0) in k,m and l,n, respectively, n = 4 and 5 for SDR-NP(D4) or SDR-HP(D4) in k,m and l,n, respectively. o, Fecal BODIPY concentrations of mice in AL-NP (n = 7 biologically independent mice), SDR-NP (n = 9) and SDR-HP (n = 9) groups after oral gavage with olive oil containing BODIPY-labeled fatty acid for 10 min to 2 h at D5. p, Representative fluorescent section images of small intestinal villi from mice in o after oral gavage for 2 h. q,r, Small intestine (q) and serum (r) BODIPY concentrations of mice in p. n = 7 and 8 biologically independent mice for AL-NP(D5) in q and r, respectively, n = 8 and 7 for SDR-NP(D5), n = 9 and 8 for SDR-HP(D5). s, Representative fluorescent section images of iWAT and eWAT from mice in o after oral gavage for 2 h. t, BODIPY concentrations of iWAT and eWAT in s (n = 9 biologically independent mice per group). u,v, Respiratory exchange ratio (RER) (u) and average RER (v) of mice as treated in a. n = 4 biologically independent mice for AL-NP, n = 6 for SDR-NP and SDR-HP. Data are presented as mean ± s.d. Statistical significance was determined by two-tailed Student’s t-test (a,b,e–h,o,q,r,t,v) and one-tailed Student’s t-test (i–n). a, *P < 0.05; b, **P < 0.01; c, ***P < 0.001; NS, not significant; except indicated, SDR-HP group versus AL-NP or SDR-NP group, SDR-NP(PF) or SDR-HF(PF) group versus SDR-HP group. Exact P values can be found in Source Data Fig. 4. Source data Full size image

Then we investigated whether food intake was involved in blocking quick fat mass accumulation after dieting by HP diet. As shown in Fig. 4b and Extended Data Fig. 6m–o, cumulative food intake of mice in the HP group significantly decreased during the refeeding period compared to that of mice in the NP groups no matter with or without dieting; however, the daily food intake after dieting in the HP group was similar to that of the NP group without dieting (Extended Data Fig. 6p–s). As shown in Fig. 2e,f and Extended Data Fig. 4e–j, refeeding with 100% food after SDR also led to a quick fat mass accumulation, so therefore the decrease of food intake during the first several days of refeeding induced by the HP diet is unlikely to be the major reason preventing quick fat mass accumulation after SDR. To further confirm whether calorie intake and/or diet composition was involved, we performed a pair-feeding experiment with NP or high-fat diet containing the same daily calorie consumed by the mice refeeding with an HP diet after SDR (Fig. 4c,d and Extended Data Fig. 7a,b). As shown in Fig. 4e and Extended Data Fig. 7c–f, pair-feeding with NP or high-fat diet after SDR induced obvious fat mass increase, which was even more severe than that in mice refeeding ad libitum with NP diet during the late refeeding period (Fig. 4e). These data show that the difference in diet composition rather than that in caloric intake has the major contribution for blocking fat mass increase after dieting by HP diet.

To further investigate how the HP diet blocks fat mass increase after SDR, we analyzed whether intestinal lipid absorption was involved. We found that both fecal TG and cecal fecal total long-chain fatty acids (LCFAs) of mice fed the HP diet after SDR significantly increased compared to the mice fed NP diet after SDR (Fig. 4f,g), suggesting that the HP diet leads to a decrease in intestinal lipid absorption. Profiling of LCFAs in cecal feces showed that refeeding after SDR reduced the levels of 15 LCFAs in all 22 detected LCFAs and the reduction of six LCFAs including the most abundant C16:0 and C18:0 induced by refeeding after SDR were blockable by an HP diet (Fig. 4h). Bile acids are known to facilitate digestion and absorption of dietary lipids53,54. Consistently, the increase of both intestinal and serum total bile acids in mice refeeding with NP diet after SDR was observed compared to those in mice fed ad libitum and the increase was repressed in mice refeeding with an HP diet after SDR (Fig. 4i,j). Both primary and secondary bile acids had a similar alteration as total bile acids in mice refeeding with NP and HP diet after SDR (Fig. 4k–n). Furthermore, gavage of BODIPY-labeled fatty acid combined with olive oil showed that fecal BODIPY levels significantly increased in the HP group (Fig. 4o). Moreover, the fluorescence intensity of villus sections from proximal jejunum in mice gavaged with BODIPY-labeled fatty acid significantly decreased in the HP group compared to that of the NP group with SDR (Fig. 4p), which is further confirmed by direct quantification of the intestinal and serum BODIPY levels (Fig. 4q,r). These data show that the decrease in intestinal lipid absorption induced by the HP diet contributes to the prevention of fat mass increase after SDR.

To further understand how decreased intestinal lipid absorption finally blocks the fat mass accumulation after SDR, we analyzed the fatty acid uptake and lipid synthesis in WAT. Gavage of BODIPY-labeled fatty acid showed that the fluorescence intensity of iWAT and eWAT sections was significantly decreased in the HP diet group compared to the mice fed NP diet after SDR (Fig. 4s), which is further confirmed by direct quantification of BODIPY levels (Fig. 4t). The results from gavage of BODIPY-labeled fatty acid demonstrate that the HP diet after SDR induces a decrease in lipid uptake in WAT. Furthermore, fatty acid and TG synthesis was significantly decreased in the iWAT of mice in the HP diet group when analyzed by high-throughput RNA sequencing (Extended Data Fig. 7g). In addition, the respiratory exchange ratio of mice fed with an HP diet after SDR was dramatically decreased (Fig. 4u,v), indicating that the HP diet increased total lipid oxidation in mice. These data demonstrate that decreased fatty acid uptake and lipid synthesis in WAT and increased total lipid oxidation induced by HP diet contribute to the prevention of fat mass increase after SDR.

Gut microbiota regulates intestinal lipid absorption after SDR

Diet is a key determinant of the gut microbiota configuration42 and microbiota can affect intestinal lipid absorption and lipid metabolism in adipose tissues49,55,56. To investigate whether microbiota are involved in the quick fat mass accumulation and increased intestinal lipid absorption induced by refeeding after SDR, we collected cecal feces of mice refed after SDR with 10%, 25% and 65% food from day 1 to day 3 and analyzed the microbiota composition by 16S rRNA gene sequencing. To assess how refeeding after SDR affects the gut microbial community structure, we investigated the α and β diversity associated with each group and compared the microbial diversity within and between communities. Principal-coordinate analysis revealed distinct clustering between microbiota from mice fed ad libitum, NP diet after SDR and HP diet after SDR (Fig. 5a). Moreover, bacterial α diversity analyzed by Shannon index was reduced after refeeding with NP diet after SDR on day 4 and was returned to normal level for the mice fed an HP diet after SDR on day 4 (Fig. 5b). Next, we calculated the percentage of bacterial taxa in each group. We observed a dramatic increment up to approximately 50% in the class Bacilli, the family Lactobacillaceae and the genus Lactobacillus during refeeding with NP diet after SDR on day 4 compared to feeding ad libitum (Fig. 5c–e); however, the relative abundance of Bacilli, Lactobacillaceae and Lactobacillus was clearly lower in mice fed an HP diet than those of mice fed a NP diet after SDR on day 4 (Fig. 5c–e). Analysis with the linear discriminant analysis (LDA) effect size (LEfSe) method revealed that the mice refeeding with NP diet after SDR was characterized by the genus Lactobacillus, which also belongs to the characterized phylum Firmicutes, class Bacilli, order Lactobacillales and family Lactobacillaceae (Fig. 5f). Songbird analysis also showed that the highest ranked genus based on the log ratios between microbiota in the SDR-NP group on day 4 and microbiota in mice fed ad libitum was Lactobacillus (Fig. 5g). These data show that refeeding after SDR significantly changes the composition of gut microbiota and induces a dramatical enrichment of Lactobacillus.

Fig. 5: Refeeding after SDR induces quick fat mass accumulation and increases intestinal lipid absorption and fatty acid uptake in WAT via alterations of microbiome especially including Lactobacillus Lam-1. a, Principal-coordinate analysis (PCoA) based on the weighted UniFrac of microbiota composition of cecal feces in mice fed ad libitum (AL) with NP diet (n = 14 biologically independent mice) or fed with NP diet (n = 7) or HP diet (n = 7) after SDR with 10%, 25% and 65% food from day 1 to day 3 at the indicated time points. Each dot represents an individual mouse. PCoA1 and PCoA2 represent the percentage of variance explained by each coordinate. P = 0.001. b, Comparison of α-diversity of microbiota in a using Shannon index (n = 14 mice for AL-NP, n = 7 for SDR-NP or SDR-HP). c,d, Class-level (c) and family-level (d) proportional abundance of microbiota in a (n = 14 mice for AL-NP, n = 7 for SDR-NP or SDR-HP). e, Genus-level proportional abundance of Lactobacillus in microbiota from a (n = 7 biologically independent mice per group). f, Cladogram generated from LEfSe and LDA score showing the most differentially significant abundant taxa enriched in microbiota from mice in a. p, phylum; c, class; o, order; f, family; g, genus. g, Songbird analysis of genera from mice in AL-NP(D0) and SDR-NP(D4) groups. The top and bottom five ranked features are highlighted with the indicated color. h, Phylogenetic tree of the isolated L. murinus Lam-1 with their relatives based on 16S rRNA gene sequences. The bar indicates sequence divergence. i, The abundance of Lam-1 determined by qPCR of 16S rDNA in cecal feces of mice as treated in a (n = 8 biologically independent mice per group). j,k, The 16S rDNA copies of Lam-1 (j) and total bacterial load (k) quantified by qPCR in cecal feces in i (n = 8 biologically independent mice per group). l,m, Effect of the indicated antibiotics on the growth of Lam-1 (n = 3 independent experiments per group and in triplicate for each experiment). PURO, puromycin; NEO, neomycin; MTZ, metronidazole; KAN, kanamycin; VAN, vancomycin; CHL, chloramphenicol; TET, tetracycline; AMP, ampicillin; PEN, penicillin. n, Experimental design of mice treated with or without penicillin given in drinking water. Arrows show the time points of feces collection, BODIPY-labeled fatty acid gavage and sampling. o,p, The abundance of Lam-1 (o) and total bacterial load (p) in feces of mice from regimens in n (n = 7 and 8 biologically independent samples for SDR on D0 and D4 respectively, n = 9 for SDR-PEN). q, Representative fluorescent section images of small intestinal villi of mice from regimens in n with or without penicillin treatment after oral gavage with olive oil containing BODIPY-labeled fatty acid for 2 h at D5. r, BODIPY concentrations of small intestine in q (n = 8 biologically independent mice for SDR, n = 9 for SDR-PEN). s, Representative fluorescent section images of iWAT and eWAT from mice in q. t, BODIPY concentrations of iWAT and eWAT in s (n = 8 mice for SDR, n = 9 for SDR-PEN). u, Penicillin treatment ameliorated fat mass accumulation induced by SDR with 10%, 25% and 65% food from day 1 to day 3 (n = 10 biologically independent mice for SDR, n = 12 for SDR-PEN). v, Daily food intake of mice in u (n = 10 mice for SDR, n = 12 for SDR-PEN). Data are presented as mean ± s.d. Statistical significance was determined by permutation multivariate analysis of variance (a), Kruskal–Wallis and Dunn’s tests (b) and two-tailed Student’s t-test (e,i–k,m,o,p,r,t–v). a, *P < 0.05; b, **P < 0.01; ***P < 0.001; NS, not significant. Exact P values can be found in Source Data Fig. 5. Source data Full size image

To investigate whether the predominant Lactobacillus bacteria with an abundance up to about 50% during refeeding after SDR contribute to the increase in intestinal lipid absorption and fatty acid uptake in WAT, we isolated and sequenced eight colonies from the cecal feces of mice in the SDR(10–25–65%) group on day 4. These colonies had the identical 16S rRNA gene sequence and phylogenetic analysis on the basis of 16S rRNA gene sequences show that Lactobacillus murinus (NR_112689) is the closest strain type to the colonies (Fig. 5h). One of the colonies was randomly picked and termed Lam-1 for further investigation. Quantification of Lam-1 by qPCR further confirmed that refeeding with a NP diet after SDR dramatically increased the abundance of Lam-1 and this increase was sufficiently suppressed when refed with an HP diet (Fig. 5i,j). In addition, the total bacterial load of cecal feces was similar in all the mice with or without SDR (Fig. 5k).

To investigate the role of gut microbiota in the quick fat mass accumulation induced by refeeding after SDR, we treated the mice with antibiotic mixture during the refeeding period after SDR (Extended Data Fig. 8a). The antibiotic-induced microbiome depletion was confirmed by gel electrophoresis of fecal DNA and quantification with qPCR (Extended Data Fig. 8b,c). Expectedly, gavage of BODIPY-labeled fatty acid showed that intestinal fatty acid absorption was significantly reduced in mice treated with an antibiotic mixture after SDR (Extended Data Fig. 8d–f). Meanwhile, fatty acid uptake in WAT was significantly decreased in mice treated with an antibiotic mixture after SDR (Extended Data Fig. 8g,h). Furthermore, treatment with an antibiotic mixture significantly ameliorated the fat mass accumulation induced by refeeding after SDR with 10%, 25% and 65% food from day 1 to day 3 (Extended Data Fig. 8i) and also repressed the increase of food intake and fat mass percentage and the decrease of lean mass percentage (Extended Data Fig. 8j–n). It has been reported that antibiotic-induced microbiome depletion alters metabolic homeostasis, including the increase of GLP-1 and decrease of luminal bile acids, suggesting the reduction of intestinal absorption of lipids57. To exclude the effect of antibiotic-induced microbiome depletion, we detected antibiotic susceptibility of Lam-1 and found that penicillin specifically inhibited the growth of Lam-1 at the concentration as low as 10 ng ml−1 (Fig. 5l,m). We next treated mice with penicillin 1 week before SDR (Fig. 5n). Penicillin treatment dramatically inhibited the growth of Lam-1 without affecting the total bacterial load (Fig. 5o,p) and significantly reduced intestinal fatty acid absorption and fatty acid uptake in iWAT of mice during refeeding after SDR (Fig. 5q–t). Moreover, penicillin treatment significantly ameliorated the fat mass accumulation induced by refeeding after SDR and also repressed the increase of fat mass percentage and the decrease of lean mass percentage without altering food intake (Fig. 5u,v and Extended Data Fig. 8o–t). These data suggest that refeeding after SDR increases fat mass accumulation, intestinal lipid absorption and fatty acid uptake in WAT via gut microbiota.

Lactobacillus increases intestinal lipid absorption and fat mass

To further investigate the effect of gut microbiota on intestinal absorption and fatty acid uptake in WAT, we transferred cecal feces from donor mice fed ad libitum or fed with NP or HP diet after SDR with 10%, 25% and 65% food from day 1 to day 3 to specific-pathogen-free (SPF) recipient mice (Extended Data Fig. 9a). As expected, intestinal lipid absorption and fatty acid uptake in eWAT of recipients transferred with microbiota from the SDR-NP group were significantly increased, which was attenuated in recipients transferred with microbiota from the SDR-HP group (Fig. 6a–e and Extended Data Fig. 9b).

Fig. 6: Gavage of Lactobacillus Lam-1 enhances intestinal lipid absorption, increases fatty acid uptake in WAT and induces fat mass accumulation. a, Fecal BODIPY concentrations of fecal microbiota transplantation (FMT)-recipient mice from regimens in Extended Data Fig. 9a after oral gavage with olive oil containing BODIPY-labeled fatty acid for 10 min to 2 h (n = 6 biologically independent mice per group). Mice received FMT from donor mice in the AL-NP, SDR-NP or SDR-HP group. AL, ad libitum. b, Representative fluorescent section images of small intestinal villi and BODIPY concentrations of small intestine from mice in a after oral gavage for 2 h (n = 6 mice per group). c, Serum BODIPY concentrations of mice in b (n = 6 mice per group). d, Representative fluorescent section images of iWAT and eWAT from mice in b. e, BODIPY concentrations of iWAT and eWAT in d (n = 6 mice per group). f, Experimental design of germ-free (GF) mice gavaged with Lactobacillus LC2W or Lactobacillus Lam-1. Arrows indicate the time points of gavage and sampling. g, Fecal BODIPY concentrations of mice from regimens in f after oral gavage with olive oil containing BODIPY-labeled fatty acid for 10 min to 2 h at D5 (n = 8 biologically independent mice per group). h, Representative fluorescent section images of small intestinal villi and BODIPY concentrations of small intestine from mice in g after oral gavage for 2 h (n = 8 mice per group). i, Serum BODIPY concentrations of mice in h (n = 8 mice per group). j, Representative fluorescent section images of iWAT and eWAT from mice in h. k, BODIPY concentrations of iWAT and eWAT in j (n = 8 mice per group). l, The weight of iWAT and eWAT in j (n = 8 mice per group). m, Schematic of the gnotobiotic (GB) mouse model illustrating the timeline of gavage with eight bacterial strains and confirmation of colonization in GF mice for subsequent LC2W or Lam-1 gavage as indicated in f. n, Fecal BODIPY concentrations of GB mice from regimens in m after oral gavage with olive oil containing BODIPY-labeled fatty acid for 10 min to 2 h at D5 (n = 8 biologically independent mice per group). o, Representative fluorescent section images of small intestinal villi and BODIPY concentrations of small intestine from mice in n after oral gavage for 2 h (n = 9 mice for GB + LC2W, n = 8 for GB + Lam-1). p, Serum BODIPY concentrations of mice in o (n = 9 mice per group). q, Representative fluorescent section images of iWAT and eWAT from mice in o. r, BODIPY concentrations of iWAT and eWAT in q (n = 9 mice per group). s, The weight of iWAT and eWAT in q (n = 9 mice per group). t, Fecal BODIPY concentrations of SPF mice gavaged with LC2W or Lam-1 after intragastric administration of olive oil containing BODIPY-labeled fatty acid for 10 min to 2 h (n = 8 biologically independent mice per group). u, Representative fluorescent section images of small intestinal villi and BODIPY concentrations of small intestine from mice in t after BODIPY gavage for 2 h (n = 8 mice per group). v, Serum BODIPY concentrations of mice in u (n = 8 mice per group). w, Representative fluorescent section images of iWAT and eWAT from mice in u. x, BODIPY concentrations of iWAT and eWAT in w (n = 8 mice per group). y, Oral gavage of Lam-1 markedly increased body fat mass (n = 9 biologically independent mice for SPF + vehicle and SPF + LC2W, n = 8 for SPF + Lam-1). z, Cumulative food intake of mice in y (n = 9 mice for SPF + vehicle and SPF + LC2W, n = 8 for SPF + Lam-1). Data are presented as mean ± s.d. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Exact P values can be found in Source Data Fig. 6. Source data Full size image

To determine whether L. murinus Lam-1 plays a causal role in the increase in intestinal lipid absorption, fatty acid uptake in WAT and fat mass accumulation, we gavaged germ-free mice with 2 × 109 colony-forming units (c.f.u.) Lam-1 or Lactobacillus paracasei LC2W for three consecutive days followed by SDR with 10%, 25% and 65% food from day 1 to day 3 (Fig. 6f). We found that Lam-1 gavage significantly increased intestinal lipid absorption and fatty acid uptake in WAT of germ-free mice during refeeding after SDR compared to LC2W gavage (Fig. 6g–k). Meanwhile, Lam-1 gavage significantly upregulated the weight of eWAT in germ-free mice during refeeding after SDR (Fig. 6l).

To further confirm the causal role for Lam-1 in upregulation of intestinal absorption, fatty acid uptake and fat mass accumulation, we used a gnotobiotic mouse model. Gnotobiotic mice were obtained from germ-free mice gavaged with eight bacterial strains representing members of the major bacterial phyla in murine gut and the colonization of bacterial strains in gnotobiotic mice was confirmed by 16S rDNA sequencing (Fig. 6m and Extended Data Fig. 9c). Then we gavaged the gnotobiotic mice with 2 × 109 c.f.u. Lam-1 or LC2W for three consecutive days followed by SDR with 10%, 25% and 65% food from day 1 to day 3. We found that Lam-1 gavage significantly increased intestinal lipid absorption and fatty acid uptake in iWAT of gnotobiotic mice during refeeding after SDR compared to LC2W gavage (Fig. 6n–r). Meanwhile, Lam-1 gavage significantly upregulated the weight of iWAT and eWAT in gnotobiotic mice during refeeding after SDR (Fig. 6s).

We next investigated the effects of Lam-1 administration on intestinal lipid absorption, fatty acid uptake in WAT and fat mass accumulation of SPF mice. Mice were gavaged orally with vehicle or 1010 c.f.u. LC2W or Lam-1 twice with an interval of 24 h. Following the last gavage, we gavaged the mice with BODIPY-labeled fatty acid. We found that Lam-1 gavage significantly increased intestinal lipid absorption and fatty acid uptake in WAT of SPF mice compared to vehicle or LC2W gavage (Fig. 6t–x and Extended Data Fig. 9d–i). Meanwhile, daily gavage of Lam-1 significantly induced fat mass accumulation in SPF mice without altering food intake (Fig. 6y,z and Extended Data Fig. 9j–m).

Collectively, the above studies indicate that Lam-1 enriched by refeeding after SDR can enhance intestinal lipid absorption, increase fatty acid uptake in WAT and induce fat mass accumulation.

Lactobacillus metabolites increase intestinal lipid absorption and fat mass

Given the effectiveness of depletion of Lam-1 by penicillin and Lam-1 administration, we sought to gain further insight to how gut microbiota orchestrates intestinal lipid absorption and fatty acid uptake in WAT during refeeding after SDR. To this end, we compared the cecal fecal non-targeted metabolomics profiles between the mice fed ad libitum and the mice fed NP and HP diets after SDR with 10%, 25% and 65% food from day 1 to day 3. As expected, refeeding with NP diet after SDR induced significant alterations in the cecal fecal metabolome compared to that in mice feeding ad libitum and these alterations were partially blocked by refeeding with HP diet after SDR (Fig. 7a). Among the metabolites in the blockable-increase group, DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), 2-hydroxyisocaproic acid (HICA), 2-hydroxy-3-methylbutyric acid (HMBA) and indole-lactic acid (ILA) are the top five significantly increased metabolites induced by refeeding with NP diet after SDR (Fig. 7a). Furthermore, the alterations of these five metabolites between different groups were further confirmed by targeted quantitative analysis using LC–MS/MS with their standards (Fig. 7b–f). Spearman’s rank correlation analysis showed that Lactobacillus was most strongly associated with cecal fecal levels of PLA, HPLA, ILA, HICA and HMBA (Fig. 7g,h). Moreover, gavage with Lactobacillus Lam-1 also significantly elevated these five metabolites in cecal feces (Fig. 7i). To further confirm whether Lam-1 produces these five metabolites, we detected these five metabolites in bacterial lysates and culture supernatant. We found that Lam-1 produced much higher amounts of PLA, HPLA, ILA, HICA and HMBA than Escherichia coli and much higher amounts of PLA, HPLA and ILA than Lactobacillus LC2W (Fig. 7j,k). These data demonstrate that refeeding after SDR induces HP-diet partially blockable alterations of cecal fecal metabolites, such as the increase of PLA, HPLA, ILA, HICA and HMBA, which can also be produced by Lam-1 and upregulated by gavage with Lam-1.

Fig. 7: Metabolites modulated by refeeding after SDR enhances intestinal lipid absorption, increases fatty acid uptake in WAT and leads to fat mass accumulation. a, Heat map of intestinal metabolites from mice fed ad libitum (AL) (n = 6 biologically independent samples) or fed with NP diet (n = 3 samples) or HP diet (n = 3 samples) after SDR with 10%, 25% and 65% food from day 1 to day 3. b–f, Concentrations of cecal fecal PLA (b), HPLA (c), ILA (d), HICA (e) and HMBA (f) from mice in a (n = 4 biologically independent samples per group). g,h, Heat map of Spearman’s rank correlation coefficients (two-sided tests) between the relative abundances of gut microbiota genera and the relative concentrations of PLA, HPLA, ILA, HICA and HMBA in cecal feces from mice in SDR-NP(D4) and AL-NP(D0) groups (g) or from mice in SDR-HP(D4) and SDR-NP(D4) groups (h). i, Concentrations of cecal fecal metabolites after oral gavage with vehicle or Lam-1 (n = 9 biologically independent samples per group). j,k, Production of PLA, HPLA, ILA, HICA and HMBA by E. coli, Lactobacillus LC2W or Lactobacillus Lam-1. E. coli was cultured in LB medium and LC2W or Lam-1 was cultured in MRS medium. Metabolites were measured in bacterial lysates (j) or culture supernatant (k) (n = 5 biologically independent samples per group). l, Fecal BODIPY concentrations of mice administrated intragastrically with water or solution containing five compounds (compds), including PLA, HPLA, ILA, HICA and HMBA after oral gavage with olive oil containing BODIPY-labeled fatty acid for 10 min to 2 h (n = 10 biologically independent mice per group). m, Representative fluorescent section images of small intestinal villi from mice in l after BODIPY gavage for 2 h. n, BODIPY concentrations of small intestine in m (n = 10 mice per group). o, Representative fluorescent section images of iWAT and eWAT from mice in m. p, BODIPY concentrations of iWAT and eWAT in (o) (n = 10 mice per group). q, Oral gavage of solution containing the five compounds markedly increased body fat mass (n = 8 biologically independent mice for water, n = 9 for compounds). r, Cumulative food intake of mice in q (n = 8 mice for water, n = 9 for compounds). s, Schematic diagram of refeeding after DR-induced obesity via microbiota and metabolite-mediated increased intestinal lipid absorption and fatty acid uptake in WAT. Data are presented as mean ± s.d. Statistical significance was determined by two-tailed Student’s t-test. * or #P < 0.05; ** or ##P < 0.01; ***P < 0.001; NS, not significant. #The SDR-NP(D4) or SDR-NP(D6) group was compared to the SDR-NP(D0) group. *SDR-HP group was compared to SDR-NP group. Exact P values can be found in Source Data Fig. 7. Source data Full size image

To investigate whether the alterations of metabolites contribute to the enhanced intestinal lipid absorption, increased fatty acid uptake in WAT and fat mass accumulation, we planned to treat mice with the top altered metabolites. Among the top five significantly increased metabolites in blockable-increase group, PLA, HPLA and ILA can be derived from ingested dietary phenylalanine, tyrosine and tryptophan by intestinal microbiota through the reductive pathway using the same enzymes58. Thus, we first investigated the effects of these three metabolites on metabolism and found that neither PLA, HPLA, ILA alone nor their mixtures had significant effect on food intake, intestinal lipid absorption and fatty acid uptake of WAT in mice (Extended Data Fig. 10a–h). In addition, gavage with HICA or HMBA also among the top five significantly increased metabolites had no significant effect (Extended Data Fig. 10i–p); however, when mice were gavaged with the mixtures of PLA, HPLA, ILA, HICA and HMBA followed with BODIPY-labeled fatty acid gavage, the fecal BODIPY levels of mice were significantly decreased without altering food intake (Fig. 7l and Extended Data Fig. 10q). Moreover, the fluorescence intensity of villus sections from proximal jejunum was significantly increased in the mice gavaged with the five metabolites compared to that of mice gavaged with water (Fig. 7m), which is further confirmed by direct quantification of the intestinal BODIPY levels (Fig. 7n). In addition, fatty acid uptake in WAT was also measured and the fluorescence intensity of iWAT and eWAT sections was significantly increased in the mice gavaged with the five metabolites (Fig. 7o), which is further confirmed by direct quantification of BODIPY levels (Fig. 7p). To exclude the potential effects of calories in the five metabolites on intestinal lipid absorption and fatty acid uptake in WAT, we treated mice with glutamic acid and glucose unchanged during refeeding after SDR using the same dose as the total five metabolites. We found that neither glutamic acid nor glucose had significant effect on intestinal lipid absorption, fatty acid uptake of WAT and food intake in mice (Extended Data Fig. 10r–v). Moreover, we found that daily gavage of the five metabolites led to significant fat mass accumulation in mice without altering food intake (Fig. 7q,r and Extended Data Fig. 10w–z). These data show that the five metabolites produced by gut microbiota, such as Lactobacillus Lam-1, can upregulate intestinal lipid absorption, enhance fatty acid uptake in WAT and induce fat mass accumulation.