Decreased nicotinamide adenine dinucleotide (NAD⁺) levels contribute to various pathologies such as ageing, diabetes, heart failure and ischemia–reperfusion injury (IRI). Nicotinamide riboside (NR) has emerged as a promising therapeutic NAD⁺ precursor due to efficient NAD⁺ elevation and was recently shown to be the only agent able to reduce cardiac IRI in models employing clinically relevant anesthesia. However, through which metabolic pathway(s) NR mediates IRI protection remains unknown. Furthermore, the influence of insulin, a known modulator of cardioprotective efficacy, on the protective effects of NR has not been investigated. Here, we used the isolated mouse heart allowing cardiac metabolic control to investigate: (1) whether NR can protect the isolated heart against IRI, (2) the metabolic pathways underlying NR-mediated protection, and (3) whether insulin abrogates NR protection. NR protection against cardiac IRI and effects on metabolic pathways employing metabolomics for determination of changes in metabolic intermediates, and ¹³C-glucose fluxomics for determination of metabolic pathway activities (glycolysis, pentose phosphate pathway (PPP) and mitochondrial/tricarboxylic acid cycle (TCA cycle) activities), were examined in isolated C57BL/6N mouse hearts perfused with either (a) glucose + fatty acids (FA) (“mild glycolysis group”), (b) lactate + pyruvate + FA (“no glycolysis group”), or (c) glucose + FA + insulin (“high glycolysis group”). NR increased cardiac NAD⁺ in all three metabolic groups. In glucose + FA perfused hearts, NR reduced IR injury, increased glycolytic intermediate phosphoenolpyruvate (PEP), TCA intermediate succinate and PPP intermediates ribose-5P (R5P) / sedoheptulose-7P (S7P), and was associated with activated glycolysis, without changes in TCA cycle or PPP activities. In the “no glycolysis” hearts, NR protection was lost, whereas NR still increased S7P. In the insulin hearts, glycolysis was largely accelerated, and NR protection abrogated. NR still increased PPP intermediates, with now high ¹³C-labeling of S7P, but NR was unable to increase metabolic pathway activities, including glycolysis. Protection by NR against IRI is only present in hearts with low glycolysis, and is associated with activation of glycolysis. When activation of glycolysis was prevented, through either examining “no glycolysis” hearts or “high glycolysis” hearts, NR protection was abolished. The data suggest that NR’s acute cardioprotective effects are mediated through glycolysis activation and are lost in the presence of insulin because of already elevated glycolysis.

烟酰胺腺嘌呤二核苷酸 (NAD ⁺ ) 水平降低会导致多种病理，如衰老、糖尿病、心力衰竭和缺血再灌注损伤 (IRI)。由于有效提高 NAD ⁺水平，烟酰胺核苷 (NR) 已成为一种有前途的治疗性 NAD ⁺前体，并且最近被证明是唯一能够在采用临床相关麻醉的模型中减少心脏 IRI 的药物。然而，NR 通过哪些代谢途径介导 IRI 保护仍不清楚。此外，尚未研究胰岛素（一种已知的心脏保护功效调节剂）对 NR 保护作用的影响。在这里，我们使用允许心脏代谢控制的离体小鼠心脏来研究：(1) NR 是否可以保护离体心脏免受 IRI，(2) NR 介导的保护的代谢途径，以及 (3) 胰岛素是否消除 NR 保护。 NR 对心脏 IRI 的保护以及对代谢途径的影响，采用代谢组学测定代谢中间体的变化，并采用¹³ C-葡萄糖通量组学测定代谢途径活性（糖酵解、磷酸戊糖途径 (PPP) 和线粒体/三羧酸循环（TCA 循环） ）活性），在用（a）葡萄糖+脂肪酸（FA）（“轻度糖酵解组”），（b）乳酸+丙酮酸+FA（“无糖酵解组”）灌注的离体C57BL/6N小鼠心脏中进行检查，或 (c) 葡萄糖 + FA + 胰岛素（“高糖酵解组”）。 NR 增加了所有三个代谢组中的心脏 NAD ⁺ 。在葡萄糖 + FA 灌注的心脏中，NR 减少了 IR 损伤，增加了糖酵解中间体磷酸烯醇丙酮酸 (PEP)、TCA 中间体琥珀酸和 PPP 中间体核糖-5P (R5P) / 景天庚酮糖-7P (S7P)，并且与激活的糖酵解相关，但不改变TCA 循环或 PPP 活动。在“无糖酵解”心脏中，NR 保护作用消失，而 NR 仍增加 S7P。在胰岛素心脏中，糖酵解大大加速，NR 保护被消除。 NR 仍会增加​​ PPP 中间体，目前S7P 的¹³ C 标记较高，但 NR 无法增加代谢途径活动，包括糖酵解。 NR 对 IRI 的保护作用仅存在于糖酵解水平较低的心脏中，并且与糖酵解的激活有关。当通过检查“无糖酵解”心脏或“高糖酵解”心脏阻止糖酵解激活时，NR 保护被废除。数据表明，NR 的急性心脏保护作用是通过糖酵解激活介导的，并且由于糖酵解已经升高而在胰岛素存在时消失。