Upregulation of colonic luminal polyamines produced by intestinal microbiota delays senescence in mice

Abstract

Prevention of quality of life (QOL) deterioration is associated with the inhibition of geriatric diseases and the regulation of brain function. However, no substance is known that prevents the aging of both body and brain. It is known that polyamine concentrations in somatic tissues (including the brain) decrease with increasing age, and polyamine-rich foods enhance longevity in yeast, worms, flies, and mice, and protect flies from age-induced memory impairment. A main source of exogenous polyamines is the intestinal lumen, where they are produced by intestinal bacteria. We found that arginine intake increased the concentration of putrescine in the colon and increased levels of spermidine and spermine in the blood. Mice orally administered with arginine in combination with the probiotic bifidobacteria LKM512 long-term showed suppressed inflammation, improved longevity, and protection from age-induced memory impairment. This study shows that intake of arginine and LKM512 may prevent aging-dependent declines in QOL via the upregulation of polyamines.

Figure 1. Screening of putrescine-upregulating substances by culture methods.

(a) PA production of a single culture of E. coli MG1655, Blautia producta JCM 1471^T, or Bacteroides thetaiotaomicron JCM 5827^T with candidate substances (GABA, Arg, Lys, Pro, fumaric acid, Asp, and Ser). Candidates that increased PA concentration more than 2-fold were used in experiments that required fecal culture. (b) PUT concentration stimulated by candidate substances with human fecal culture. Data are expressed as relative ratio that calculate PUT concentration from 0 h to 1 h. Results of each fecal specimen are shown in Supplementary Fig. S1A. Data are represented as mean ± SD **p < 0.01, ***p < 0.001. (c) Dose-dependent effect of Arg on PUT production with human fecal culture.

Figure 2. Effects of Arg administration on putrescine concentration in rodents.

(a) Fecal PA concentration after oral administration of Arg in ICR mice (left) and SD rats (right) (n = 8) *p < 0.05, **p < 0.01, ***p < 0.001 (vs. control = 0 mg). (b) Influences of oral administration of antibiotics on PA production due to Arg and effects of ornithine substitution on PA production in ICR mice (n = 8). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (vs. Arg 0.6 mg/g body). nt: not tested. (c) Polyamine concentration in the colonic lumen and tissue after oral Arg administration in SD rats (n = 4). (d) Changes of blood PA concentration after oral Arg administration in rats with a jugular catheter (n = 5). *p < 0.05 (vs. pre-administration). (e) Fecal PA concentration after injection of Arg through a proximal colon catheter (n = 5). *p < 0.05, **p < 0.01 (vs. control = 0 mg).

Figure 3. Detection of stable isotope-labeled putrescine derived from ¹³C₆,¹⁵N₄ Arg in colonic content.

Both stable isotope-labeled putrescine (m/z 128, 171, and 361) and unlabeled putrescine (m/z 126, 166, and 355) were detected within the colonic lumen.

Figure 4. Effects of 6-month oral administration of Arg, LKM512, or Arg & LKM512 mix in aging ICR mice.

The test sample was administered 3 times a week, starting when the mice were 12 months old. Data were obtained from 7 mice in the Arg and LKM512 groups, 6 mice in the Arg & LKM512 mix group (a mouse died in the study period), and 4 mice in the control group (3 mice died in the study period). (a) Serum inflammatory cytokine concentrations *p < 0.05, **p < 0.01. (b) SMP-30 content in liver tissue (right: western blot, left: immunohistochemical staining). (c) A dendrogram of fecal microbiota of mice after a 6-month treatment based on the T-RFLP pattern. (d) Bacterial PUT synthesis pathway from an Arg precursor. Pathway 1: decarboxylation of ornithine generated from Arg hydrolysis. Pathway 2: direct conversion of agmatine generated from Arg decarboxylation, catalyzed by agmatine ureohydrolase. Pathway 3: indirect conversion of agmatine generated from Arg decarboxylation via N-carbamoylputrescine, catalyzed by agmatine deiminase/iminohydrolase and N-carbamoylputrescine amidohydrolase (e) Comparison of the proportion of PUT synthesis-related genes detected from both the Arg & LKM512 mix group and the control group by fecal metagenome analysis.

Figure 5. Long-term oral administration of Arg & LKM512 mix delays aging in ICR mice.

The Arg & LKM512 mix was administered 3 times a week, starting when the mice were aged 14 months (female Arg & LKM512 mix group: n = 62, female control group: n = 66, male Arg & LKM512 mix group: n = 10, male control group: n = 10). *p < 0.05, **p < 0.01. (a) Kaplan-Meier survival curves. (b) Differences in body weight and chow intake between the Arg & LKM512 mix group and control group. (c) Change in escape latency. (d) Time spent in four quadrants in probe test. *p < 0.05 (Student's t-test), †p < 0.05 (paired t-test).