Abstract
The AMPK-Sirt1 pathway is an important regulator of energy metabolism and therefore a potential target for prevention and therapy of metabolic diseases. We recently demonstrated leucine and its metabolite β-hydroxy-β-methylbutyrate (HMB) to synergize with low-dose resveratrol (200 nM) to activate sirtuin signaling and stimulate energy metabolism. Here we show that leucine exerts a direct effect on Sirt1 kinetics, reducing its Km for NAD+ by >50% and enabling low doses of resveratrol to further activate the enzyme (p = 0.012). To test which structure elements of resveratrol are necessary for synergy, we assessed potential synergy of structurally similar and dissimilar polyphenols as well as other compounds converging on the same pathways with leucine using fatty acid oxidation (FAO) as screening tool. Dose-response curves for FAO were constructed and the highest non-effective dose (typically 1–10 nM) was used with either leucine (0.5 mM) or HMB (5 µM) to treat adipocytes and myotubes for 24 h. Significant synergy was detected for stilbenes with FAO increase in adipocytes by 60–70% (p<0.05) and in myotubes >2000% (p<0.01). Sirt1 and AMPK activities were stimulated by ∼65% (p<0.001) and ∼50% (p<0.03), respectively. Similarly, hydroxycinnamic acids and derivatives (chlorogenic, cinnamic, and ferulic acids) combined with leucine/HMB increased FAO (300–1300%, p<0.01), AMPK activity (50–150%, p<0.01), and Sirt1 activity (∼70%, p<0.001). In contrast, more complex polyphenol structures, such as ellagic acid and epigallocatechin gallate required higher concentrations (>1 µM) and exhibited little or no synergy. Thus, the six-carbon ring structure bound to a carboxylic group seems to be a necessary element for leucine/HMB synergy with other stilbenes and hydroxycinnamic acids to stimulate AMPK/Sirt1 dependent FAO; these effects occur at concentrations that produce no independent effects and are readily achievable via oral administration.
Highlights
AMP-activated protein kinase (AMPK) and the sirtuins Sirt1 and Sirt3 are well-known key sensors of energy status and regulators of glucose and lipid metabolism [1,2,3]
There is evidence for an indirect Sirt1 activation mediated by inhibiting cAMP phosphodiesterase, which results in upregulation of AMPK and a subsequent increase in NAD+ levels [11]
To evaluate fatty acid oxidation, measured as the oxygen consumption rate (OCR) with the Seahorse XF analyzer, as an appropriate method of screening, we tested the effects of Resv and Leu, individually and in combination, as well as of AMPK and Sirt1 inhibitors in this system
Summary
AMP-activated protein kinase (AMPK) and the sirtuins Sirt and Sirt are well-known key sensors of energy status and regulators of glucose and lipid metabolism [1,2,3]. There is evidence for an indirect Sirt activation mediated by inhibiting cAMP phosphodiesterase, which results in upregulation of AMPK and a subsequent increase in NAD+ levels [11]. This was shown to be the case only at high concentrations (50 mM) that are not achieved in vivo, while lower concentrations lead to direct Sirt activation [12]. These different modes of action may explain reports of Resv’s dose- and time- dependent effects, which lead to different outcomes in cell and animal studies. Studies in humans are very limited and results from cell and animals studies are not readily translated, since, due to the low bioavailability of Resv, plasma concentrations achieved with oral supplementation are much lower than those used in vitro
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