Supplemental information

Supplemental information of a research article "Effect of chronic administration with human thioredoxin-1 transplastomic lettuce on diabetic mice"<https://doi.org/10.1002/fsn3.2391

コウサンカ タンパク デ アル チオレドキシン ワ サンカ ストレス ニ ヨル マウス ハイ ノ ハッセイ イジョウ オ フセグ

京都大学0048新制・課程博士博士(医学)甲第9728号医博第2520号新制||医||805(附属図書館)UT51-2002-J510京都大学大学院医学研究科生理系専攻(主査)教授 西川 伸一, 教授 中辻 憲夫, 教授 塩田 浩平学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry.

NAD+ is mainly synthesized from nicotinamide (Nam) by the rate-limiting enzyme Nam phosphoribosyltransferase (Nampt) and degraded to Nam by NAD+-degrading enzymes in mammals. Numerous studies report that tissue NAD+ levels decrease during aging and age-related diseases and suggest that NAD+ replenishment promotes healthy aging. Although increased expression of Nampt might be a promising intervention for healthy aging, forced expression of Nampt gene, inducing more than 10-fold increases in the enzyme protein level, has been reported to elevate NAD+ levels only 40-60% in mammalian cells. Mechanisms underlying the limited increases in NAD+ levels remain to be determined. Here we show that Nampt is inhibited in cells and that enhanced expression of Nampt activates NAD+ breakdown. Combined with the measurement of each cell's volume, we determined absolute values (μM/h) of the rates of NAD+ synthesis (RS) and breakdown (RB) using a flux assay with a 2H (D)-labeled Nam, together with the absolute NAD+ concentrations in various mammalian cells including primary cultured cardiomyocytes under the physiological conditions and investigated the relations among total cellular Nampt activity, RS, RB, and the NAD+ concentration. NAD+ concentration was maintained within a narrow range (400-700 μM) in the cells. RS was much smaller than the total Nampt activity, indicating that NAD+ synthesis from Nam in the cells is suppressed. Forced expression of Nampt leading to 6-fold increase in total Nampt activity induced only a 1.6-fold increase in cellular NAD+ concentration. Under the conditions, RS increased by 2-fold, while 2-fold increase in RB was also observed. The small increase in cellular NAD+ concentration is likely due to both inhibited increase in the NAD+ synthesis and the activation of its breakdown. Our findings suggest that cellular NAD+ concentrations do not vary dramatically by the physiological fluctuation of Nampt expression and show the tight link between the NAD+ synthesis and its breakdown

Supplemental information

SCOPE: Human thioredoxin‐1 (hTrx‐1) is a defensive protein induced by various stresses and exerts antioxidative and anti‐inflammatory effects. Previously, we described a transplastomic lettuce overexpressing hTrx‐1 that exerts a protective effect against oxidative damage in a pancreatic β‐cell line. In this study, we treated diabetic mice (Akita mice) with exogenous hTrx‐1 and evaluated the effects. METHODS AND RESULTS: Treatment with drinking water and single applications of exogenous hTrx‐1 did not influence the feeding, drinking behavior, body weight, blood glucose, or glycosylated hemoglobin (HbA(1c)) levels in Akita mice. However, chronic administration of a 10% hTrx‐1 lettuce‐containing diet was associated with a significant reduction from the baseline of HbA(1c) levels compared with mice fed a wild‐type lettuce‐containing diet. It also resulted in an increased number of goblet cells in the small intestine, indicating that mucus was synthesized and secreted. CONCLUSION: Our results revealed that the administration of an hTrx‐1 lettuce‐containing diet improves the baseline level of HbA(1c) in Akita mice. This effect is mediated through goblet cell proliferation and possibly related to protection against postprandial hyperglycemia by mucus, which results in the improvement of blood glucose control. These findings suggest that the hTrx‐1 lettuce may be a useful tool for the continuous antioxidative and antidiabetic efficacies of the hTrx‐1 protein