Phlorizin ameliorates obesity-associated endotoxemia and insulin resistance in high-fat diet-fed mice by targeting the gut microbiota and intestinal barrier integrity

Abstract

Phlorizin (PHZ) is one of phytonutrients in apples that contributes to the health-promoting effect implicated by the saying, 'an apple a day keeps the doctor away'. PHZ was firstly identified as a competitive inhibitor of sodium-glucose co-transporters-2 (SGLT2); however, its low bioavailability makes it hard to fully explain its pharmacological mechanisms. This study aimed to investigate the ameliorating effect of PHZ on high-fat diet (HFD)-induced obesity via modulating the "gut microbiota-barrier axis". Firstly, C57BL/6 J mice were fed a normal chow diet (NCD) or HFD coadministered with or without PHZ for 12 weeks. Our results showed that PHZ supplementation significantly reduced HFD-induced body weight gain (P < .001), alleviated metabolic disorders (MDs) like insulin resistance (P < .001) and elevation of serum lipopolysaccharides (LPS) (P < .001), attenuated HFD-induced gut microbiota alterations, enhanced short-chain fatty acids (SCFAs) production (P < .001), and inhibited fecal LPS production (P < .001). To investigate the role of the fecal microbiota in the observed beneficial effects, a fecal microbiota transplantation (FMT) experiment was performed by transplanting the feces of the four groups of mice (as donor mice) daily collected from the fourth week to a new batch of acclimatized HFD-fed mice. Our results confirmed that feeding the gut contents of the PHZ-modulated mice could attenuate HFD-induced MDs, accompanied by enhanced glucagon-like peptide 2 (GLP-2) secretion (P < .001) and restoration of HFD-induced damage in the gut epithelial barrier. This study has provided evidence that the "gut microbiota-barrier axis" was an alternative target for the anti-obesity effect of PHZ. This work has also provided an explanation for the high efficacy of PHZ despite the low bioavailability, and PHZ holds great potential to be developed as a functional food ingredient.

Keywords: Phlorizin (PHZ); barrier integrity; glucagon-like peptide 2 (GLP-2); gut microbiota; obesity; short-chain fatty acids (SCFAs).

Figure 1.

Phlorizin (PHZ) attenuated high-fat diet (HFD)-induced obesity. (a) Changes in the body weight of mice over 12 weeks. At week 12, multiple obesity-related parameters were recorded for the four groups of mice receiving normal chow diet (NCD), NCD with PHZ (NCD+PHZ), HFD, and HFD with PHZ (HFD+PHZ), respectively. The parameters included: (b) food intake; (c) energy intake; (d) fat mass; (e) weights of different organs; morphological observations of the (f) liver and (g) epididymis fat; (h) hematoxylin and eosin staining of epididymis fat; (i) adipocyte size of epididymis fat; levels of serum (j) lipopolysaccharide (LPS) and (k) glucagon-like peptide-1 (GLP-1); (l) fasting blood insulin; (m) fasting glucose; (n) homeostasis model assessment (HOMA)-insulin resistance (IR) index; (o) HOMA-insulin sensitivity (IS) index; (p) oral glucose tolerance test; (q) insulin tolerance test. Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze statistical differences; NS for P > .05, *P < .05, **P < .01, and ***P < .001

Figure 2.

Phlorizin (PHZ) attenuated high-fat diet (HFD)-induced microbial and metabolic dybiosis. (a) Principal components analysis (PCA) score plot and (b) hierarchical clustering of fecal microbiota of the four groups of mice receiving normal chow diet (NCD), NCD with PHZ (NCD+PHZ), HFD, and HFD with PHZ (HFD+PHZ), respectively. (c) Chao 1 index representing the α-diversity of the gut microbiota. (d) Phylum-level distribution of fecal microbiota; (e, f) relative abundance of the phyla Firmicutes and Bacteroidetes; (g) ratio between the relative abundance of Firmicutes and Bacteroidetes; (h-m, o-p) relative abundance of identified differential abundant bacterial groups at different taxonomic levels; (n) Genus-level distribution of fecal microbiota; (q) fecal lipopolysaccharide (LPS); (r) total short-chain fatty acids (SCFAs). Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze statistical differences; NS for P > .05, **P < .01 and ***P < .001

Figure 3.

Phlorizin (PHZ) attenuated high-fat diet (HFD)-induced damage of the intestinal barrier. (a) Hematoxylin and eosin staining and (b) periodic acid-Schiff staining of intestinal tissue sections of the four groups of mice receiving normal chow diet (NCD), NCD with PHZ (NCD+PHZ), HFD, and HFD with PHZ (HFD+PHZ), respectively. Samples were taken at 12^th week. The blue marks in (a) indicate intestinal wall thickness; the arrows in (b) indicate the stained goblet cells. Various parameters indicating the integrity of intestinal barrier were analyzed at 12^th week, including (c) serum glucagon-like peptide-2 (GLP-2) level; (d) villus height; (e) intestinal wall thickness; (f) number of goblet cells; and (g) intestinal mucus thickness. One-way ANOVA was used to analyze statistical differences, *P < .05, **P < .01, and ***for P < .001. Data are expressed as mean ± standard deviation. (h) Pearson correlation analysis was performed between various fecal metabolites and indicators of gut epithelial integrity. The color scale represents the strength of correlation, ranging from 0.5 (strong positive correlation) to −0.5 (strong negative correlation)

Figure 4.

Transplantation of feces from mice fed phlorizin (PHZ) attenuated high-fat diet (HFD)-induced obesity and damage of intestinal barrier. (a) Chromatograms of PHZ and phloretin (PHT) in the feces of the four groups of donor mice fed normal chow diet (NCD), NCD with PHZ (NCD+PHZ), HFD, and HFD with PHZ (HFD+PHZ). The four groups of recipient mice (maintained on HFD) that underwent fecal microbiota transplantation were designated as “NCD→HFD”, “NCD+PHZ→HFD”, “HFD→HFD”, and “HFD+PHZ→HFD”, respectively. (b) Changes in body weight over eight weeks. A number of obesity- and gut epithelial barrier integrity-related parameters were recorded at eighth week, including (c) the weights of epididymis fat and liver; (d) hematoxylin and eosin (H & E) staining of epididymis fat; (e) adipocyte size of epididymis fat; (f) liver morphology; (g, h) fasting blood glucose and insulin levels; (i-k) serum levels of glucagon-like peptide-2 (GLP-2), diamine oxidase (DAO), and D-lactate; (l, m) H&E and AB-PAS staining of intestinal tissue sections. The blue marks in (l) indicate intestinal wall thickness; the arrows in (m) indicate the stained goblet cells. Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze statistical differences; NS for P > .05, *P < .05, **P < .01, and ***P < .001

Figure 5.

Schematic diagram showing the possible mechanisms of anti-metabolic disorders (MDs) effects of phlorizin (PHZ). Other than the primary target of sodium-glucose co-transporters-2 (SGLT2), the ‘gut microbiota-barrier axis’, including the gut microbiota and the gut barrier, served as alternative targets for the beneficial action of PHZ. GLP: glucagon-like peptide; LPS: lipopolysaccharide; SCFAs: short-chain fatty acids

Figure 6.

Experimental design. PHZ: phlorizin; PHT: phloretin; GLP: glucagon-like peptide; LPS: lipopolysaccharide; SCFAs: short-chain fatty acids; H&E: hematoxylin and eosin; AB-PAS: periodic acid-Schiff; NCD: normal chow diet; HFD: high-fat diet. FMT: fecal microbiota transplantation