Old-age-induced obesity reversed by a methionine-deficient diet or oral administration of recombinant methioninase-producing Escherichia coli in C57BL/6 mice

Increase of mouse body weight with age

Mouse body weight increased with age. The mean body weight in mice aged 1, 2 months was 25.4 g; 2–6 months was 29.4 g (p = 0.0109 with respect to 1–2 months); 6–12 months was 41.1 g (p < 0.0001 with respect to 2–6 months); and 12–18 months was 45.5 g (p = 0.0248 with respect to 6–12 months). (Figure 1).

Figure 1. Body weight increase with age in C57BL/6 male mice.

Obesity was reversed by oral administration. rMETase-producing E. coli JM109

Figure 2A shows each group’s percent body weight change for 14 days. In mice fed non-recombinant E. coli JM109 cells (control group), mouse body weight did not change significantly during 14 days. The mice administered recombinant E. coli JM109-rMETase (E. coli JM109-rMETase group) decreased their mean body weight from 43.8 g to 40.7 g by day 15. This 3.1 g body weight loss is significantly higher than that of the control group (p = 0.0325). In the mice fed a methionine- and choline-deficient diet (methionine-deficient diet group), body-weight loss was 5.6 g (p = 0.0001). Mouse body weight continued to decrease until 15 days on the low-methionine diet. (Figure 2B).

Figure 2. (A) Percent body weight change from study days 1–15. (B) Body weight change from baseline in each group on day 15.

After day 15, the body weight of the methionine-deficient-diet group continued to decrease until week 15 with a total average loss of 26.3 g. (Figure 3) Fifteen weeks after the beginning of the methionine-deficient diet, mouse body weight stabilized at 20 grams.

Figure 3. Long-term body weight change in mice on the methionine-deficient diet.

Methionine level

On day 15, the mean methionine level of each group was as follows: control group: 108.4 μM; E. coli JM109-rMETase group: 71.3 μM; methionine-deficient diet group: 34.0 μM. Both the methionine-deficient diet group and the E. coli JM109-rMETase-treated group showed significantly lower levels of methionine compared to the control group (Figure 4A). On day 29, the mean methionine level of each group was 81.2, 59.1, and 15.5 μM, respectively (Figure 4B). There was a negative correlation between the methionine level and negative body-weight change on day 29, R² = 0.685, (Figure 5).

Figure 4. Blood methionine level at day 15 (A) and day 29 (B).

Figure 5. The correlation between methionine level and body weight change on day 29.

Stool culture of E. coli JM109-rMETase group

E. coli JM109-rMETase was detected in the E. coli JM109-rMETase group stool on day 15 and day 22 indicating incorporation in the microbiome.

Mice

C57BL/6 male mice (AntiCancer Inc., San Diego, CA, USA) aged 1-18 months were used in the present study. The mice were housed in a barrier facility with a HEPA-filtered rack under typical light/dark cycles of 12 hours. Mice were given an autoclaved laboratory rodent meal before the beginning of this study. The AntiCancer Institutional Animal Care and Use Committee’s ethics committee granted approval in accordance with National Institutes of Health Guide Assurance Number 3873-1. All experiments adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines [19].

Culture of E. coli JM109-rMETase

The host strain for the expression of rMETase was E. coli JM109. The P. putida rMETase gene was previously cloned into E. coli JM109 using the plasmid pATG3131, which also contains the tetracycline (TC) resistance gene [14, 15]. The generated E. coli JM109-rMETase was pre-cultured in 5 ml of liquid Luria-Bertani (LB) medium with TC (32 g/ml) for eight hours at 37°C. The pre-culture broth was transferred overnight to 400 ml culture medium containing 32 g/ml TC. To promote the expression of rMETase, isopropyl—D-thiogalactopyranoside (IPTG) was administered at a final concentration of 0.3 mM for 4 hours at 28°C. The concentration of E. coli JM109-rMETase was adjusted to 10¹¹ CFU/mL using phosphate-buffered saline (PBS) and 20% glycerin. E. coli JM109-rMETase was stored at −80°C until administration to mice. Non-recombinant E. coli JM109 competent cells were generated in the same manner as E. coli JM109-rMETase, except for the addition of TC and IPTG to the LB medium.

Study design

Spontaneous body-weight gain was observed in C57BL/6 mice for up to 18 months. Fifteen mice weighing more than 40 g and aged 12 to 18 months were chosen and divided into three groups of five mice each, with similar mean weight in each group: Group 1: normal diet gavaged with non-recombinant E. coli JM109 competent cells (10¹⁰/100 μL), treated twice daily (9 AM and 5 PM by oral gavage) for 14 days: Group 2: Normal diet supplemented with E. coli JM109-rMETase cells (10¹⁰/100 μL), treated by gavage twice daily (9 AM and 5 PM) for 14 days. Group 3: Mice on a methionine-deficient and choline-diet without treatment for 21 weeks. The normal diet contained 0.5% methionine, while the methionine-deficient diet was depleted of methionine, homocysteine, and choline.

IPTG (10 mM) and TC (0.5 g/L) were added to the drinking water of the mice to induce E. coli JM109-rMETase to produce rMETase and prevent plasmid shedding only in group 2 treated with E. coli JM109-rMETase [15]. Body weight was measured every two days until day 15, then every seven days until 21 weeks.

Blood and stool collection

Mouse blood was obtained through tail hemorrhage at 9 to 10 o’clock in the morning on days 15 and 29. Only the mice that received E. coli JM109-rMETase had their stool collected in the mornings of days 15, 22, and 29.

Mouse stool culture and screening for E. coli JM109-rMETase in the stool

The stool was diluted 1:10 by weight with PBS, and then mechanically disrupted. Large debris was pelletized by brief centrifugation at 200 × g. Subsequently, 100 μL of the supernatant was plated onto LB agar with 32 μg/mL TC and incubated at 37°C overnight. The screening of E. coli JM109-rMETase in the stool was performed using modified M9 agar as described previously [17].

Determination of plasma methionine level

The plasma concentration of methionine was measured using precolumn derivatization, followed by high-performance liquid chromatography separation (HPLC) based on a previously described method [20].

Statistical analysis

GraphPad Prism 9.4.0 was used to perform all statistical analyses (GraphPad Software, Inc., San Diego). Tukey-Kramer was used for the parametric test of group comparison. All data are represented by the mean and standard deviation. p-Values less than 0.05 were considered significant.