Probiotics feeding prevents HFD-induced metabolic dysfunction and physical function decline in older mice. Older adults are more susceptible to obesity- and type 2 diabetes–related metabolic dysfunctions like glucose intolerance, insulin resistance, hepatic steatosis, and inflammation in adipose tissues upon high calorie/fat intake (34–37). Here, we investigated whether our probiotic cocktail can protect such high-fat diet–induced (HFD-induced) dysfunctions in older mice. Interestingly, 10 weeks of probiotic cocktail feeding significantly increased blood glucose clearance in oral glucose tolerance test (OGTT) (Figure 1A) and improved insulin sensitivity upon exogenous insulin administration during insulin tolerance test (ITT) (Figure 1B) in HFD-fed older mice compared with their age- and sex-matched controls. These data suggest that probiotic feeding prevented HFD-induced glucose metabolism dysfunctions in older mice. However, no significant differences were observed on body weight and weight gain, food and water intake, or energy expenditure in probiotic-fed group compared with their controls (Supplemental Figure 1, A–G; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.132055DS1). This indicates that the probiotics cocktail was well tolerated and enhanced metabolic functions of older mice. Interestingly, fat accumulation in liver (hepatic steatosis) was significantly attenuated, and crown-like structures in white adipose tissue (WAT), an inflammation marker, were significantly lower in probiotic-fed older mice than in controls (Figure 1, C and D), indicating that probiotic feeding reduced fat accumulation and inflammation in metabolic organs like liver and WAT.

Figure 1 Probiotics feeding prevents HFD-induced metabolic dysfunctions in older mice. (A and B) Ten weeks of probiotics improved glucose tolerance and enhanced insulin sensitivity in older obese mice, measured by oral glucose tolerance test (A) and insulin tolerance test (B) (n = 6 in control and n = 8 in probiotics groups; *P < 0.05, 2-way ANOVA). (C) Representative images of H&E staining of liver (upper panels) showing reduced fat accumulation and white adipose tissue (WAT; lower panels) showing reduced adipocyte size, along with reduced inflammation (indicated by crown-like structures; red arrows) in probiotics fed mice (n = 8) compared with their controls (n = 6). (D) Crown-like structures are graphed. (E) Probiotic-fed older obese mice (n = 8) exhibited higher physical function presented as walking speed compared with their age- and sex-matched HFD-fed controls (n = 6). Values are mean of n = 6–8 mice in each group, and data are shown as mean ± SEM. *P < 0.05, and ***P < 0.001 by 2-way ANOVA with Bonferroni’s correction (A and B) and Student’s t test (D and E).

Obese older adults face higher decline in physical function, such as reduced walking speed that is commonly associated with poor health outcomes and increased mortality in older adults (38–40). Interestingly, probiotic-fed obese older mice had higher walking speed than controls (Figure 1E), suggesting that probiotic therapy attenuated physical function decline in older obese mice. Altogether, these results indicate that probiotic therapy prevented HFD-induced metabolic derangements like glucose intolerance, insulin resistance, hepatic steatosis, and inflammation in WAT and improved physical function of older mice.

Probiotic therapy beneficially modulates gut microbiota in obese older mice. The primary action of probiotics is posited through modulating the gut microbiota, and we found that feeding this human-origin probiotic cocktail significantly changed microbial composition in the gut of older mice (Figure 2). Reduced microbial diversity measured by α-diversity (microbial diversity within the sample) and β-diversity (microbial diversity among the samples) are known indicators of dysbiosis (41), and interestingly, feeding this probiotic cocktail enhanced microbial diversity, as indicated by increased Shannon index (α-diversity) and by a significantly distinct clustering of β-diversity observed in principal coordinate analysis (PCoA) (Figure 2, A and B). Other α-diversity indices like phylogenetic diversity (PD) whole tree, Chao1, and number of operational taxonomic units (OTUs) remain significantly unchanged in probiotic-fed mice and their control mice, but trends were toward higher α-diversity in probiotic-fed mice compared with controls (Supplemental Figure 2, A–C). The abundance of phylum Firmicutes, family Rumminoccocaceae, and an unidentified family of order Clostridiales was significantly increased, while abundance of phylum Verrucomicrobia and families Verrucomicrobiaceae and Erysipelotrichaceae was decreased in probiotic-fed mice compared with controls (Figure 2, C and D). Specifically, probiotic feeding promoted the abundance of Clostridiales;f_;g_, Oscilliospira, Allbacullum, Rosiburia, Desulfovibrio, and Dorea, while it suppressed the population of Akkermansia, S24-7, Peptococcacea;g_rc4-4, Lachnospiraceae;g_, Lactococcus, Ruminococcus, SMB53, Coprobacillus, and Lactobacillus (Figure 2E). Similar bacterial phyla, families, and genera appeared during our Linear discriminatory analysis effect size (LEfSe) analysis (Supplemental Figure 2, D and E). Interestingly, among top 10 significantly changed bacterial species due to probiotics feeding, the abundance of Akkermansia muciniphila, Peptococcus niger, and Ruminicoccus gnavus significantly decreased, while that of Clostridium histolyticum (C. histolyticum), C. thermosuccinogenes, Roseburia faecis, Enterococcus lactis, Bacteroides salanitronis, and Lactobacillus rhamnosus was significantly increased in probiotic-fed obese older mice compared with their controls (Figure 2, F–N). These results demonstrate that older HFD-fed mice that received the probiotic cocktail developed a significantly distinct gut microbiota signature — enriched with beneficial commensals — that was associated with improvement in the metabolic health of older obese mice.

Figure 2 Probiotic therapy beneficially modulates gut microbiome in older obese mice. (A–E) Gut microbiome signature in terms of β-diversity (A), α-diversity (Shannon index) (B), and abundance of major phyla (C), families (D), and genera (E) were significantly changed in probiotic-treated HFD-fed older mice (n = 5) compared with their controls (n = 5). (F–N) Specifically, probiotic therapy decreased Akkermansia muciniphila (F), Peptococcus niger (G), and Ruminicoccus gnavus (H) and increased C. histolyticum (I), C. thermosuccinogenes (J), Roseburia faecis (K), Enterococcus lactis (L), Bacteroides salanitronis (M), and Lactobacillus rhamnosus (N). Values are mean of n = 5 in each group, and data are shown as mean ± SEM. *P < 0.05; **P < 0.01, and ***P < 0.001 by PERMANOVA (A), unpaired 2-tailed Student’s t test (F–N), and 1-way ANOVA (B–E).

Probiotics feeding reduces inflammation and leaky gut markers in HFD-fed older mice. Low-grade inflammation is a major risk factor of metabolic dysfunctions, poor health, and high mortality in older adults (4, 42). Herein, we found that the probiotics feeding significantly decreased the expression of proinflammatory markers like IL-6, TNF-α, and IL-1β in LPS-treated primary macrophages isolated from the peritoneal cavities of older mice compared with their controls (Figure 3, A–C), indicating that the probiotic feeding suppressed the inflammatory responses in the mucosal lymphatic macrophages present in peritoneal cavity. Similarly, the expression of IL-6, TNF-α, and IL-1β was decreased, while antiinflammatory markers like IL-10 and TGF-β expression was increased in the colon tissues of probiotic-fed mice (Figure 3, D–H), suggesting that probiotic feeding also suppresses the inflammation in the intestinal milieu. These results demonstrate that probiotic therapy reduced inflammation in intestinal tissues and distant immune cells like peritoneal lymphatic macrophages.

Figure 3 Probiotics treatment reduces inflammation in peritoneal macrophages and intestine of older obese mice. (A–C) LPS-induced inflammatory response in terms of mRNA expression of IL-6 (A), TNF-α (B), and IL-1β (C) was reduced in primary macrophages isolated from peritoneal cavity of older HFD-fed mice treated with probiotics (n = 8) compared with their controls (n = 6). (D–H) In addition, the expression of proinflammatory markers such as IL-6 (D), TNF-α (E), and IL-1β (F) were decreased, while antiinflammatory genes like IL-10 (G) and TGF-β (H) mRNA expressions were increased in the colon of probiotic-fed older obese mice (n = 8) compared with their controls (n = 6). (I and J) In addition, systemic leaky gut markers such as LPS binding protein (LBP) (I) and soluble CD14 (J) were reduced in the serum of probiotic-fed older obese mice (n = 7) compared with their controls (n = 6). Values are mean of n = 6–8 in each group, and data are shown as mean ± SEM. *P < 0.05; **P < 0.01, ***P < 0.001 by Student t-test (A–J).

Increased inflammation in older adults is often associated with increased leaky gut indicated by system markers such as LPS binding protein (LBP) and soluble CD14 (sCD14) in circulation/blood that are linked with poor health outcomes (3, 5). Interestingly, we found that the feeding of our human-origin probiotic cocktail significantly reduced LBP levels in the serum of older mice compared with their controls (Figure 3I). The sCD14 levels were also decreased in the serum of probiotic-fed older mice but did not achieve statistical significance (Figure 3J). These results indicated that the probiotics feeding reduces leaky gut markers that are linked with reduced inflammation in intestine and peritoneal macrophages.

Probiotics feeding enhances expression of tight junction proteins in obese older mice. Abnormalities in the gut epithelium, specifically the downregulation of tight junction proteins, are associated with compromised intestinal epithelial permeability (leaky gut) and increased inflammation in obesity and aging (3, 41). We demonstrated that the probiotic therapy significantly increased the mRNA expression of tight junction proteins such as Zo1 and Ocln in the intestinal tissues of older obese mice (Figure 4, A and B). The protein expression of Zo1 increased, while marginal increase with no statistical significance in Ocln protein was seen (Figure 4, C and D). This suggests that probiotic therapy improved the intestinal epithelial integrity that is linked to reduced inflammation in local intestinal tissues, as well as systemically, as corroborated by the decrease in peritoneal macrophages. Interestingly, global and unbiased gene expression analysis by RNA sequencing (RNAseq) revealed that the probiotics feeding significantly changed the expression of a large number of genes and pathways. Specifically, probiotics feeding significantly upregulated around 856 genes — while downregulating around 1053 genes in the gut of older mice compared with their controls (Figure 4E) — that were very distinctly clustered among these groups (Figure 4F). Furthermore, pathway analyses revealed that the cell adhesion molecules (CAMs) and cytokine pathways were the top 2 largely affected pathways in the intestinal tissues of probiotic-fed obese older mice in comparison with their controls (Figure 4G). These results demonstrate that the probiotic therapy enhanced cell adhesion pathways and particularly increased tight junction proteins in the intestine of older obese mice that were associated with decreased inflammation.

Figure 4 Probiotics treatment increases expression of tight junctions in the intestine of older obese mice. (A and B) The mRNA expression of tight junction proteins like Zonulin-1 (Zo1) (A) and Occludin (Ocln) (B) were significantly increased in colon of probiotics fed older mice (n = 8) compared with their controls (n = 6). (C and D) Western blot analysis shows that Zo1 protein expression was significantly increased, while Ocln showed marginal increase in the colon tissues of probiotic-treated older mice (n = 7) compared with their controls (n = 6). (E and F) Global gene expression using RNAseq analysis revealed that probiotic feeding significantly increased around 856 genes while it decreased 1053 genes that were distinctly clustered in the probiotic-treated (n = 7) group versus controls (n = 6). (G) Pathway analysis of deferentially expressed genes (DEGs) shows that cell adhesion and cytokine (immune) pathways were more affected by probiotics treatment compared with their controls. Values are mean of n = 6–7 each group, and data are shown as mean ± SEM. **P < 0.01 and ***P < 0.001. Student t test (A, B, D) and random forest analysis (E) were used, as well as hierarchical clustering between samples using hclust, with diagrams drawn with ggplot2 (F) and differential expression of genes (DEGs) (G) were completed using R programs.

Probiotics primarily act on intestinal tight junctions instead of immune cells. We demonstrate that the probiotic therapy improved metabolic functions and gut microbiota that are associated with reduced inflammation and increased expression of tight junction proteins; however, it is not known whether (a) probiotic-modulated gut microbiota primarily act on intestinal epithelium by improving tight junctions to reduce inflammation or vice versa or (b) these changes in tight junctions and inflammation are parallel events. We used a coculture system of human intestinal epithelial cells (Caco-2; in upper chamber) and human macrophages (THP-1–differentiated macrophages; in lower chamber) to test the effect of probiotics cecal conditioned media (CCM), which was prepared from probiotic-fed mouse cecal contents compared with control CCM. When Caco-2 cells were exposed to CCM of probiotic-fed mice, we observed significantly reduced intestinal epithelial permeability measures like transepithelial electrical resistance (TEER) and FITC-dextran diffusion in the Caco-2 monolayer compared with the CCM of control mice (Figure 5, A and B). However, no significant changes in TEER and FITC diffusion of the Caco-2 monolayer were observed when cocultured macrophages were exposed to both CCM (Figure 5, E and F). Similarly, the expression of tight junction proteins (Zo1 and Ocln) mRNA was significantly increased in Caco-2 monolayer treated with CCM of probiotic-fed mice compared with their controls (Figure 5, C and D), while no significant changes were observed in these measures in Caco-2 cells cocultured with CCM-treated macrophages (Figure 5, G and H). These results demonstrate that the probiotic-modulated gut microbiota primarily act on intestinal epithelial cells to increase the expression of tight junction proteins, resulting in decreased epithelial permeability, which in turn reduced inflammation in local tissues and peripheral immune cells.

Figure 5 Probiotics primarily act on intestinal tight junctions versus immune cells. (A–C) Treatment of Caco-2 cell monolayers (cultured with THP1 cells) with cecal conditioned media (CCM) from probiotic-fed mice shows significantly less changes in transepithelial electrical resistance (TEER) (A) with reduced FITC-dextran (4 kDa) (B) and increased Zo1 (C) and Ocln (D) mRNA and protein expressions compared with control CCM–treated cells. (E–H) No significant changes were observed in these measures when cocultured THP-1 cells were treated with CCM prepared from probiotics and control mouse cecal contents. Values are mean of 2–3 repeated experiments done in triplicate, and CCM was prepared from cecal contents of n = 6 controls and n = 7 probiotic-treated mice. Data are shown as mean ± SEM. **P < 0.01 ***P < 0.001 by ANOVA with Bonferroni’s corrections and Student’s t test.

Probiotics feeding significantly modulates metabolites and increases taurine abundance in the gut of HFD-fed older mice. To determine the missing link by which probiotic-modulated gut microbiota improves intestinal tight junctions and inflammation in obese older mice, we tested the hypothesis that gut microbiota–derived metabolites may be involved in these improvements. Therefore, we performed untargeted-unbiased global metabolomics in the feces of these mice (43). Interestingly, the metabolites from probiotic-fed mice were clustered distinctly from their controls (Figure 6, A and B). In addition, the volcano plot suggests that major metabolites like taurine, glucose, and total bile acids (Figure 6, E–G) (44), as well as glycine, acetate, isoleucine, phenylalanine, and tyrosine, were significantly abundant in the gut of probiotic-fed compared with control older obese mice (Figure 6C). Furthermore, the heatmap in Figure 6D also shows that various metabolites in terms of fold change are significantly distinct in probiotic-fed versus control mice. Altogether, these results indicate that probiotics feeding significantly changed metabolites in the gut of older mice.

Figure 6 Probiotic therapy modulates gut metabolome and increases taurine production by enhancing bile salt hydrolase (BSH) activity in the gut of older HFD-fed mice. (A–C) Untargeted-unbiased metabolomics analyses show that the production of distinct metabolites shown by principal component analysis (PCA) (A), group clustering (B), and fold change abundance enrichment (C) were significantly changed in probiotic-fed old mice (n = 5) compared with their controls (n = 5). (D) Similarly, metabolite heatmap shows differential clusters that are significantly changed in probiotic-fed older mice feces compared with controls. (E–I) Abundance of taurine (E) and total bile acids (F), glucose (G), butyrate (H) and propionate (I) was significantly increased in the feces of probiotic-fed older mice (n = 5) compared with their controls (n = 5). (H) Probiotics feeding significantly enhanced bile salt hydrolase (BSH) activity in the gut of older mice. Values are mean of n = 5 control and n = 5 probiotics group, and data are shown as mean ± SEM. **P < 0.01 and ***P < 0.001. PLS-tool box in MatLab (A and B) and Welch t-test (E–J) were used.

A pathway analysis plotted using pathway enrichment analysis and pathway impact values further indicated that the major Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) pathways significantly affected after probiotics feeding were taurine and hypotaurine metabolism (Supplemental Figure 3). The abundance of taurine, total bile acids, glucose, butyrate, and propionate were significantly increased in the gut of probiotic-fed older mice compared with their controls (Figure 6, E–I). The highest increase (fold change, 3.9) in taurine levels in the feces of probiotic-fed older mice (Figure 6E) suggested that taurine is linked with improvements in intestinal tight junctions, inflammation, and metabolic, physical, and cognitive functions in older mice fed with HFD. However, the mechanism of how taurine levels increased in the feces of probiotic-fed mice is not known. Taurine is a modified amino acid produced in the liver and released by conjugation to bile acids (bile salts) such as taurocholate in the gut through bile juice (45, 46). These taurine-carrying bile salts are deconjugated by a bacterial enzyme, bile salt hydrolase (BSH) (47). Therefore, we tested a hypothesis that probiotics feeding may have increased BSH activity in the gut microbiota, resulting in increased deconjugation of taurine from bile acids like taurocholate. Interestingly, the BSH activity was significantly increased in the feces of probiotic-fed older mice compared with their controls (Figure 6J), suggesting that probiotics changed the gut microbiota in a way that increased BSH activity, which resulted in higher deconjugation of tauro-bile salts to release more taurine in the gut. Most of the strains in our probiotics cocktail show BSH activity (Supplemental Figure 4, A and B), suggesting that feeding of the probiotic cocktail increased BSH activity in the gut of older mice, which may lead to increased deconjugation of free taurine from bile salts.

Taurine restores intestinal integrity and improves health indicators in C. elegans. To further determine whether increased taurine levels in the gut contributes to increasing tight junctions, thus reducing intestinal epithelial permeability, we supplemented taurine (200 μM) in the CCM and incubated it with Caco-2 cell’s monolayer. Interestingly, taurine supplementation significantly reduced decline in TEER and increased the expression of Zo1 and Ocln mRNA (Figure 7, A–C, and Supplemental Figure 5), indicating that taurine increases intestinal epithelial barrier integrity to reduce gut permeability by increasing the expression of tight junction proteins. Furthermore, to determine the impact of taurine feeding on life-span in Caenorhabditis elegans (C. elegans), we showed that taurine feeding extended the life span of C. elegans by > 2 days, which is equivalent of > 10–12 years of human age (Figure 7D), suggesting that taurine exhibits antiaging effects. In addition, taurine feeding also reduced fat accumulation and intestinal permeability (measured by using Smurf assay; ref. 48) (Supplemental Figure 6) in older C. elegans (Figure 7, E–G). This further reveals that increased taurine in the gut is beneficial to ameliorate aging-related dysfunctions like adiposity and leaky gut, which in turn improves healthy life-span.