Mulberry Leaf Supplements Effecting Anti-Inflammatory Genes and Improving Obesity in Elderly Overweight Dogs

Abstract

Overweight and obesity, associated with various health complications, refer to abnormal or excessive fat accumulation conditions that harm health. Like humans, obesity is a growing problem in dogs, which may increase the risk of serious diseases such as diabetes and cancer. Mulberry leaf has shown potential anti-obesity and anti-diabetes effects in several studies. Our research studied the impact of mulberry leaf supplements in healthy old overweight dogs for 12 weeks. Blood and fecal samples were collected from the dogs before and after treatment for different analyses, including whole transcriptome and gut microbiome analysis. The Body Condition Score (BCS) and blood glucose levels were significantly decreased in all mulberry treatment groups, which justifies the anti-obesity effect of mulberry leaf in dogs. Throughout the whole transcriptome study, the downregulation of PTX3 and upregulation of PDCD-1, TNFRSF1B, RUNX3, and TICAM1 genes in the high mulberry group were found, which have been associated with anti-inflammatory effects in the literature. It may be an essential gene expression mechanism responsible for the anti-inflammatory and, subsequently, anti-obesity effects associated with mulberry leaf treatment, as confirmed by real-time polymerase chain reaction analysis. In microbiome analysis, Papillibacter cinnamivorans, related to the Mediterranean diet, which may cause anti-inflammatory effects, were abundant in the same treatment group. Further studies may be required to establish the gene expression mechanism and role of abundant bacteria in the anti-obesity effect of mulberry supplements in dogs. Overall, we propose mulberry leaves as a portion of food supplements for improving blood glucose levels and the anti-inflammation of blood in companion dogs.

Keywords: Papillibacter cinnamivorans; anti-inflammation; companion; mulberry leaf; obesity; overweight.

Figure 1

Comparison of body condition score (BCS) (a), body weight (BW) (b), glucose (c), cortisol (d), leptin (e), adiponectin (f) levels in obese dogs with low-dose (Mulberry_40) or high-dose (Mulberry_100) of mulberry leaf treatment for 12 weeks. * of BCS (a) shows the p-value of <0.05 vs. Mulberry_0 group. All data are presented as mean ± SD. * shows p < 0.05 and ** is p < 0.01. Mulberry_0: normal diet group supplemented with 40 mg/kg/day of maltodextrin (placebo); Mulberry_40: normal diet group supplemented with 40 mg/kg/day of the mulberry leaf; Mulberry_100: normal diet group supplemented with 100 mg/kg/day of the mulberry leaf.

Figure 2

Number of differentially expressed genes before and after treatment. Mulberry_0: normal diet group supplemented with 40 mg/kg/day of maltodextrin (placebo); Mulberry_40: normal diet group supplemented with 40 mg/kg/day of the mulberry leaf; Mulberry_100: normal diet group supplemented with 100 mg/kg/day of the mulberry leaf.

Figure 3

Quantitative expressions of diabetes-related genes between the before and after groups. (a) PTX3, (b) PDCD1, (c) TNFRSF1B, (d) RUNX3, (e) TICAM1, and (f) CDKN1A. All data are presented as mean ± SEM, and tests were performed in three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The red lines expressed GeneCounts between the before and after groups. Mulberry_0: normal diet group supplemented with 40 mg/kg/day of maltodextrin (placebo); Mulberry_40: normal diet group supplemented with 40 mg/kg/day of the mulberry leaf; Mulberry_100: normal diet group supplemented with 100 mg/kg/day of the mulberry leaf.

Figure 4

Differential abundance of taxa in the Mulberry_0, Mulberry_40, and Mulberry_100 groups. (a) Bar plot depicting differentially abundant bacterial taxa according to LDA. (b) Cladogram. Mulberry_0: normal diet group supplemented with 40 mg/kg/day of maltodextrin (placebo); Mulberry_40: normal diet group supplemented with 40 mg/kg/day of the mulberry leaf; Mulberry_100: normal diet group supplemented with 100 mg/kg/day of the mulberry leaf.

Figure 5

Visualization of correlation matrix among microbiome and obesity-related factors.

Figure 6

Heatmap showing the occurrence of KOs, which were differential in the comparison of the Mulberry_100 group vs. the Mulberry_0 group. Mulberry_0: normal diet group supplemented with 40 mg/kg/day of maltodextrin (placebo); Mulberry_100: normal diet group supplemented with 100 mg/kg/day of the mulberry leaf.