Background

We have previously shown leucine (Leu) to activate Sirt1 by lowering its K_M for NAD⁺, thereby amplifying the effects of other sirtuin activators and improving insulin sensitivity. Metformin (Met) converges on this pathway both indirectly (via AMPK) and by direct activation of Sirt1, and we recently found Leu to synergize with Met to improve insulin sensitivity and glycemic control while achieving ~ 80% dose-reduction in diet-induced obese mice. Accordingly, we sought here to define the mechanism of this interaction.

Methods

Muscle cells C2C12 and liver cells HepG2 were used to test the effect of Met–Leu on Sirt1 activation. Caenorhabditis elegans was used for glucose utilization and life span studies.

Results

Leu (0.5 mmol/L) + Met (50–100 μmol/L) synergistically activated Sirt1 (p < 0.001) at low (≤ 100 μmol/L) NAD⁺ levels while Met exerted no independent effect. This was associated with an increase in AMPK and ACC, phosphorylation, and increased fatty acid oxidation, which was prevented by AMPK or Sirt inhibition or silencing. Met–Leu also increased P-IRS1/IRS1 and P-AKT/AKT and in insulin-independent glucose disposal in myotubes (~ 50%, p < 0.002) evident within 30 min as well as a 60% reduction in insulin EC₅₀. In addition, in HepG2 liver cells nuclear CREB regulated transcription coactivator 2 (CRTC2) protein expression and phosphorylation of glycogen synthase was decreased, while glycogen synthase kinase phosphorylation was increased indicating decreased gluconeogenesis and glycogen synthesis. We utilized C. elegans to assess the metabolic consequences of this interaction. Exposure to high glucose impaired glucose utilization and shortened life span by ~ 25%, while addition of Leu + Met to high glucose worms increased median and maximal life span by 29 and 15%, respectively (p = 0.023), restored normal glucose utilization and increased fat oxidation ~ two-fold (p < 0.005), while metformin exerted no independent effect at any concentration (0.1–0.5 mmol/L).

Conclusion

Thus, Leu and Met synergize to enable Sirt1 activation at low NAD⁺ concentrations (typical of energy replete states). Sirt1 and AMPK activations are required for Met–Leu's full action, which result in improvements in energy metabolism and insulin sensitivity.

中文翻译：

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亮氨酸-二甲双胍联合激活AMPK / Sirt1途径可增加骨骼肌的胰岛素敏感性，并刺激葡萄糖和脂质代谢，并延长秀丽隐杆线虫的寿命

背景

先前我们已经证明亮氨酸（Leu）通过降低其NAD ^+的K _M来激活Sirt1 ，从而放大其他瑟土因激活剂的作用并改善胰岛素敏感性。二甲双胍（Met）通过间接激活（通过AMPK）和直接激活Sirt1收敛于该途径，我们最近发现Leu与Met协同作用，以改善胰岛素敏感性和血糖控制，同时在饮食诱导的肥胖症中实现约80％的剂量减少老鼠。因此，我们在这里寻求定义这种相互作用的机制。

方法

肌肉细胞C2C12和肝细胞HepG2用于测试Met-Leu对Sirt1激活的影响。秀丽隐杆线虫被用于葡萄糖利用和寿命研究。

结果

Leu（0.5 mmol / L）+ Met（50-100μmol/ L） 在低（≤100μmol/ L）NAD ⁺水平下协同激活Sirt1（p <0.001），而Met没有独立作用。这与AMPK和ACC的增加，磷酸化和脂肪酸氧化的增加有关，这可以通过AMPK或Sirt抑制或沉默来防止。Met–Leu还增加了P-IRS1 / IRS1和P-AKT / AKT，并且在 30分钟内明显减少了肌管中胰岛素非依赖性的葡萄糖处置（〜50％，p <0.002），并且胰岛素EC ₅₀降低了60％。此外，在HepG2肝细胞中，核CREB调节的转录共激活因子2（CRTC2）蛋白表达和糖原合酶的磷酸化降低，而糖原合酶激酶的磷酸化增加，表明糖异生和糖原合成减少。我们利用秀丽隐杆线虫评估这种相互作用的代谢后果。暴露于高葡萄糖会损害葡萄糖利用率，并使寿命缩短约25％，而向高葡萄糖蠕虫中添加Leu + Met可使中位数和最大寿命分别增加29％和15％（p  = 0.023），恢复正常的葡萄糖利用率和脂肪氧化增加约两倍（p  <0.005），而二甲双胍在任何浓度（0.1–0.5 mmol / L）下均无独立作用。

结论

因此，Leu和Met协同作用以在低NAD ⁺浓度（典型的能量充足状态）下激活Sirt1 。Met-Leu的完整动作需要Sirt1和AMPK激活，从而改善能量代谢和胰岛素敏感性。