Elsevier

Phytomedicine

Volume 106, November 2022, 154423
Phytomedicine

Original Article
Laurolitsine ameliorates type 2 diabetes by regulating the hepatic LKB1-AMPK pathway and gut microbiota

https://doi.org/10.1016/j.phymed.2022.154423Get rights and content

Abstract

Background

Type 2 diabetes mellitus (DM) is a highly prevalent chronic metabolic disease. Effective antidiabetic drugs are needed to improve and expand the available treatments. Using the ob/ob diabetic mouse model, we previously demonstrated that the alkaloid-rich extract from Litsea glutinosa bark (CG) has potent antidiabetic effects and that laurolitsine (LL) is the richest alkaloid in CG.

Purpose

We conducted a systematic investigation of the antidiabetic effects and potential mechanisms of LL in vitro and in vivo.

Methods

The antidiabetic effects of LL and its mechanisms of action were explored in HL-7702 hepatocytes in vitro and in db/db mice in vivo by a series of experiments, including cellular toxicity analysis, glucose consumption analysis, serum/liver biochemical analysis, pathological examinations, Western blots, RNA-seq analysis, and gut microbiota analysis.

Results

LL stimulated glucose consumption and activated AMP-activated protein kinase (AMPK) without inducing lactic acid production or cytotoxicity in vitro. LL had potent antidiabetic effects with hypoglycemic activity in vivo. It improved insulin resistance, glucose tolerance and lipid metabolism; protected liver, renal and pancreatic functions; and promoted weight loss in db/db mice. Transcriptomic analysis suggested that the antidiabetic effects of LL involved the regulation of mitochondrial oxidative phosphorylation. We further demonstrated that LL effectively activated the hepatic liver kinase B1 (LKB1)/AMPK pathway by regulating the ADP/ATP ratio. Simultaneously, LL significantly modulated the gut microbial community, specifically decreasing the abundances of Mucispirillum schaedleri and Anaerotruncus_sp_G3_2012, which might also contribute to its antidiabetic effects.

Conclusion

These results suggest that LL is a promising antidiabetic drug candidate that may improve glucolipid metabolism via modulation of the hepatic LKB1/AMPK pathway and the gut microbiota.

Introduction

Type 2 diabetes mellitus (DM) is characterized by high blood glucose level added to a group of metabolic disorders. It is one of the most prevalent health problems, affecting a total of 537 million patients worldwide (Sun et al., 2022). Hyperglycemia and hyperlipidemia are the main clinical manifestations of DM, usually caused by pancreatic islet β-cells being unable to secrete sufficient amounts of insulin to match the progressing severity of insulin resistance (IR) (Loria et al., 2013). Glucolipid metabolism and IR in diabetes are lengthy and complex processes that mainly occur in the liver, muscles, and adipose tissues. Although drugs such as metformin, rosiglitazone, and acarbose are widely used for the control of blood glucose, they do not meet all clinical needs, and new antidiabetic drugs are still needed to expand and improve the available treatment options.

Natural products are an important source of new antidiabetic drugs. Classic examples include galegine and phlorizin, which have been isolated from plants (Ríos et al., 2015). In traditional Chinese medicine (TCM), DM has been treated with extracts from plants or other natural resources. Extracts of TCMs such as Gynostemma pentaphyllum, Coptis chinensis, and Salvia miltiorrhiza showed significant clinical effects in treating DM (Wang et al., 2018). Importantly, natural products obtained from TCMs, such as berberine and mangiferin, are very attractive antidiabetic drug candidates (Wang et al., 2019). The leaves and bark of Litsea glutinosa (Lour.) C.B.Rob. (Lauraceae) are traditionally used to treat diabetes and other diseases in China (Wu et al., 2017; Zhang et al., 2018). In our previous study, the alkaloid-rich extract from Litsea glutinosa bark (CG) was found to have antidiabetic effects. Furthermore, eight alkaloids were identified in the CG, and laurolitsine (LL) was the most abundant (Zhang et al., 2018). However, there have been no reports regarding the antidiabetic activities of LL, indicating a need for systematic investigation of its effects and mechanisms of action.

In the present study, we evaluated its antidiabetic effects in vitro using HL-7702 cells and in vivo using db/db diabetic mice, an established animal model of DM (Sharma et al., 2003). We also investigated the potential mechanisms underlying the antidiabetic effects of LL.

Section snippets

Materials

The human liver HL-7702 cell line was purchased from the Institute of Cell Biology of the Chinese Academy of Sciences (Shanghai, China). RPMI-1640 medium, fetal bovine serum (FBS), trypsin, antibiotics, and phosphate buffer solution were purchased from Gibco-Invitrogen (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from KeyGEN Biotech Co., Ltd. (Nanjing, China). The cell and tissue triglyceride (TG) assay kit was purchased from Applygen

LL shows no cytotoxicity in HL-7702 hepatocytes and potently stimulates glucose consumption

The MTT assay showed that LL with a purity of 99.5% (Fig. 1) was not toxic to HL-7702 hepatocytes at concentrations up to 320 μM (Fig. 2A). Treatment with LL (2.5 μM) for 24 h significantly stimulated glucose consumption in HL-7702 hepatocytes compared with the vehicle control (6.99 ± 0.57 mM vs. 5.84 ± 0.59 mM, p < 0.05). The efficiency of LL at 5 μM was comparable to that of 2 mM metformin (Fig. 2B).

LL potently alleviates diabetic symptoms in db/db mice

Compared to C57BL/KsJ mice, diabetic db/db mice showed typical diabetic symptoms, such as

Discussion

The leaves and bark of L. glutinosa are traditional medicines in Hainan Province of China and are traditionally used to treat diabetes. Our previous study found that CG has antidiabetic effects and that its key component was LL (Zhang et al., 2018). In this work, we demonstrated the antidiabetic effects of LL and explored its potential mechanism via a systematic investigation including biochemical and molecular analyses, RNA-seq assays and metagenomics.

Obesity, polydipsia and increased food

Conclusion

The present study demonstrated that LL could stimulate glucose consumption without inducing lactic acid production in vitro. Meanwhile, it exhibited potent antidiabetic effects, including hypoglycemic activity, weight loss, alleviation of insulin resistance, improvement of lipid metabolism, and protection of liver, kidney and pancreatic islet functions in vivo. A potential mechanism of this antidiabetic activity may be related to the decreasing abundances of M. schaedleri and

CRediT authorship contribution statement

Zhang Yong and Wang Ruiqi wrote this paper draft. Zhang xiaopo, Wu chongming and Lu weiying corrected the draft. Zhang Yong, Zhang Xiaopo supervised the experimentators. Zhang Yong, Wang Ruiqi, Yang Yanan, Maning, Zhou Zhi, Tan Yinfeng, Li Yiying and Dong Lin performed the experiments. All data were generated inhouse, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Competing Interest

All authors report no conflict of interest.

Acknowledgment

This work was financially supported by Hainan Provincial Natural Science Foundation of China (2019RC208), and the National Natural Science Foundation of China (81760628), and Project Supported by Hainan Province Clinical Medical Center (QWYH202175).

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    Authors contributed equally to this paper.

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