Original ArticleHernandezine, a natural herbal alkaloid, ameliorates type 2 diabetes by activating AMPK in two mouse models
Graphical abstract
Introduction
Type 2 diabetes mellitus (T2DM) is a pandemic metabolic disorder characterized by deregulated glucose and lipid metabolism (Perreault et al., 2021). Many key metabolic organs, including skeletal muscle and liver, become less responsive to insulin, leading to compensatory hyperinsulinemia (Czech, 2017). Patients with T2DM also have a much higher risk of suffering from cardiovascular diseases (Lu et al., 2019). Therefore, it is crucial to develop strategies that involve metabolism correction as well as cardiovascular risk reduction (Lim et al., 2011).
AMP-activated protein kinase (AMPK), a heterotrimeric enzyme comprising α (catalytic) and β/γ (regulatory) polypeptides (Salt and Hardie, 2017), is a key regulator of energy metabolism. During energy stress, AMPK is activated by AMP to phosphorylate other key factors that restore energy homeostasis (Hardie et al., 2011). Mechanistically, AMP binding causes a two- to three-fold increase in allosteric activation of AMPK (Woods et al., 2003, 2005). Both ADP and AMP can promote phosphorylation of Thr-172 on AMPK by the LKB1 and CaMKKβ kinases, increasing the kinase activity of AMPK (Langendorf and Kemp, 2015; Wu et al., 2013). ADP and AMP also protect Thr-172 against dephosphorylation (O'Neill, 2013; Ross et al., 2016). Activation of AMPK resembles the response to energy shortage caused by physical activity and caloric restriction, and reverses a series of metabolic abnormalities, including insulin resistance, obesity, hypertension, and altered plasma lipids (Musi and Goodyear, 2003; Lin and Hardie, 2018; Oakhill et al., 2011). Therefore, AMPK activation has long been considered a therapeutic target in T2DM (Hardie, 2013; Xiao et al., 2013).
Several pharmacological compounds that increase AMPK activity directly or indirectly have been developed to treat T2DM (Esquejo et al., 2018; Salatto et al., 2017). For example, compounds PF-739 and MK-8722 bind directly to the “allosteric drug and metabolite” (ADaM) site (Myers et al., 2017; Cokorinos et al., 2017) between the α and β subunits, which results in strong activation of AMPK complexes. However, these two compounds failed in preclinical trials, with PF-739 showing a lack of long-term hypoglycemic effect, and MK-8722 causing cardiac hypertrophy (Steinberg and Carling, 2019). Alternatively, plant-derived natural products such as resveratrol and berberine, which are widely used in traditional herbal medicine, can activate AMPK indirectly by inhibiting mitochondrial respiration (Hardie, 2013). These compounds are beneficial in terms of long-term safety, but may be limited in their bioavailability and effectiveness (Liu et al., 2016; Walle et al., 2004). Hernandezine (HER) is an alkaloid isolated from a traditional Chinese medicinal herb known as Thalictrum (Hsiao et al., 2016). HER is known as a Ca2+ channel antagonist (Low et al., 1996; Leung et al., 1996) and an inducer of autophagic cell death in drug-resistant cancers possibly via direct activation of AMPK (Li et al., 2017; Law et al., 2016). However, the mechanism by which HER activates AMPK is still unclear and whether HER could target AMPK to treat T2D is largely unknown. Herein, we report that HER strongly activated AMPK by increasing its phosphorylation level, effectively alleviated T2DM. In two T2DM mouse models, db/db and DIO, HER increased glucose disposal capacity and insulin sensitivity and, therefore, prevented the increases over time in blood glucose, blood lipids and body weight. At the tissue level, HER increased glucose uptake in skeletal muscle and reduced hepatic lipid synthesis, but did not induce apparent cardiac hypertrophy. Therefore, we provide a new model to explain the HER-mediated AMPK activation, and propose that HER would be a potential anti-T2DM therapeutic via targeting AMPK signaling.
Section snippets
Dephosphorylation of AMPK
HER (purity ≥ 98%, chemical structure as shown in Fig. 1A) synthesized from our laboratory (Patent Disclosure No. CN110964024A). Fully phosphorylated human AMPK α1β1γ1 (CT001-H0907B, SinoBiological, China), human AMPK α2β2γ1 (CT006-H0907B, SinoBiological, China) and the presence or absence of 10, 20 µM HER or 150 µM AMP with buffer (40 mM HEPES, 0.5 mM DTT and 2.5 mM MgCl2) was preincubated for 20 min at 30 °C, then incubated presence or absence PP2Cα (12984-H08E, SinoBiological, China) for
HER inhibits dephosphorylation of pAMPK in vitro and increases pAMPK in cells
To test whether HER (Fig. 1A) could activate AMPK directly, we obtained a recombinant isoform of human AMPK (AMPKα1β1γ1 and AMPKα2β2γ1) and measured its kinase activity with HER using the specific AMPK substrate SAMS peptide (Zhang et al., 2013a, 2013b; Steneberg et al., 2018). Surprisingly, unlike AMP, HER failed to activate AMPKα1β1γ1 isoform directly (Fig. 1B), although a previous study claimed that the kinase activity of AMPKα1β1γ1 could be detected using a general kinase substrate (
Discussion
One of the great threats to future health is chronic energy imbalance, which can increase the risks of obesity and T2DM (Roden and Shulman, 2019). Intense research in recent years has revealed critical roles played by AMPK in regulating an ever-expanding array of biological pathways in lipid and glucose metabolism, making it a very attractive target for drug discovery (Garcia and Shaw, 2017). In this study we showed that HER, a natural product, is a strong activator for AMPK, and therefore
Conclusions
In conclusion, we demonstrated that the natural product HER potently activated AMPK signaling in cells and tissues by preventing the dephosphorylation of pAMPK. Oral treatment with HER exhibited substantial antidiabetic and antiobesity effects with low risk of causing cardiac hypertrophy in two T2DM mouse models, making it a potential antidiabetic therapeutic for future development.
Authors contributions
J.B. designed experiments, researched and analyzed data, and wrote the manuscript. S.Z. performed the cell experiments. J.J.C. and Z.G.M performed animal experiments. S.Z. and H.B.S. analyzed and interpreted data. Z.G.M. synthetized HER. W.L.S. and H.L. reviewed and edited the manuscript. H.L. were the guarantors of this work, had full access to all data in the study, and take responsibility for the integrity of the data and the accuracy of the data analysis. All data were generated in-house,
Declaration of Competing Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
We thank Dr. Jingya Li of Shanghai Institute of Materia Medica for the advice for this research, Core Facility of Basic Medical Sciences. Shanghai Jiao Tong University School of Medicine for offering guides on glucose intake in vitro. Michelle Kahmeyer-Gabbe, Ph.D., from Liwen Bianji (Edanz) for editing the English text of a draft of this manuscript. This work was supported by Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (GZ2017007) and National
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These authors contributed equally to this work.