3-Bromopyruvate-induced glycolysis inhibition impacts larval growth and development and carbohydrate homeostasis in fall webworm, Hyphantria cunea Drury
Graphical abstract
Introduction
Glycolysis, the primary dissimilatory pathway in all living organisms, is the first step in the breakdown of glucose to extract energy for cellular metabolism (Bolaños et al., 2010; Kumari, 2018). As one of the primary pathways of carbohydrate metabolism, glycolysis provides the substrates for energy production via the formation of adenosine triphosphate (ATP) and substrates for storage pathways of glycogenesis and lipogenesis as well (Bhagavan and Ha, 2015). In animal cells, when under normoxic conditions, the final product of glycolysis is pyruvate (PA), which is converted to acetyl-coenzyme A (acetyl-CoA) in the mitochondria, where it is fully oxidized to CO2 through the tricarboxylic acid (TCA) cycle (Bhagavan and Ha, 2015). However, under hypoxic or anoxic status, the mitochondrial respiratory chain is impaired, the rate of glycolysis increases, so that PA, which is not metabolized further in the mitochondria, is converted into lactate at the expense of NADH(H+) oxidation (Aragonés et al., 2009). As is known, the enzymes involved in regulating glycolysis are hexokinase (HK), phosphoglucoisomerase (PGM), phosphofructokinase (PFK), aldolase (ALDO), triose phosphate isomerase (TIM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), enolase and pyruvate kinase (PK) (Bommer et al., 2020; Ghanavat et al., 2020). Definitely, the regulation of glycolysis flux is extremely complicated, which involved in various factors and signaling pathways, such as p53, hypoxia inducible factor (HIF), oncogene c-Myc, AMP-activated protein kinase (AMPK), insulin/mTOR signaling pathway, phosphatidylinositol 3-kinase (PI3K) and Akt pathway (Herzig and Shaw, 2017; Kishton et al., 2016; Mulukutla et al., 2016; Wu et al., 1994; Wu and Wei, 2012). Given the critical functions in regulating carbohydrate metabolism, insulin secretion, and ATP generation in various mammalian cells, glycolysis had been widely studied in pathogenesis correlated with diabetes, tumorigenesis, and dysmetabolic syndrome in recent years (Abad et al., 2020; Guo et al., 2012; Li et al., 2016; Stienstra and Netea, 2018; Zhang et al., 2018).
Although glycolysis is regarded as the simplest and most well-known pathway of nutrient metabolism, and much evidence has increasingly proved that glycolysis plays critical roles in a wide variety of biological functions from the perspective of integrative physiology (Guo et al., 2012; Leong et al., 2003; Orang et al., 2019; Salmina et al., 2015). In mammals, glycolysis is critically involved in the integrative regulation of glucose homeostasis, and it is tied closely to a variety of physiological events, including glucose production, insulin secretion, glycogen synthesis, alteration of inflammatory responses (Huo et al., 2010; Rossetti and Giaccari, 1990; Wu et al., 2005). Furthermore, increased glycolysis is the main source of energy supply in cancer cells that use this metabolic pathway for ATP generation. Therefore, regulations of glycolysis related genes (both at the transcriptional levels and phosphorylation levels) have been widely studied in control of malignant tumor cell survival and cancer therapy in recent decades (Abbaszadeh et al., 2020; Chen and Russo, 2012; Gatenby and Gillies, 2007; Zhang et al., 2018). Conversely, few studies focused on the roles of glycolysis in regulating insect growth and development. Furthermore, as the insect blood sugar, trehalose is not only the substrate of glycolysis but also the initial substrate for chitin synthesis (Muthukrishnan et al., 2012). However, little is known about whether and how glycolysis inhibition impacts trehalose homeostasis and chitin synthesis in insects.
Chitin is widely found in the exoskeleton and the peritrophic matrix (PM) of insects in the predominant form of a polysaccharide, whose biosynthesis, metabolism, and modification are intimately coupled with the growth and development, and metamorphosis of insects (Zhu et al., 2016). Besides providing the principal energy source for living organisms, carbohydrates (involved in trehalose and glucose) also contribute to the primary substrates for chitin synthesis in insects (Chippendale, 1978; Muthukrishnan et al., 2012; Thompson, 1998). Therefore, the interference of carbohydrate metabolism affects insect growth and development, metamorphosis, as well as chitin synthesis directly or indirectly (Baki et al., 2018; Tang et al., 2010; Zhang et al., 2020). It should be emphasized that regulation of carbohydrate metabolism in insects is extremely intricate, which is harmoniously governed by multiple signaling pathways, involved in insulin, juvenile hormone (JH), 20-hydroxyecdysone (20E) signaling pathway (Baki et al., 2018; Hou et al., 2015; Keshan et al., 2017; Suzuki et al., 2011). The chitin biosynthetic pathway has been well described in previous reports (Merzendorfer, 2006; Muthukrishnan et al., 2012). Furthermore, transcriptional regulation of genes in the chitin synthesis pathway has been extensively studied in recent decades (Yao et al., 2010; Zhao et al., 2018). In particular, many advances have been obtained in the regulation of chitin synthesis by two major hormones, steroid hormone and JH, which are closely contacted with insect molting and metamorphosis (Dubrovsky, 2005; Liu et al., 2019; Riddiford and Ashburner, 1991; Riddiford and Truman, 1993). The molecular regulatory mechanisms of chitin synthesis are being revealed gradually. However, whether genes in the glycolysis pathway are involved in regulating chitin synthesis remains largely unknown.
As a typical glycolytic inhibitor, 3-bromopyruvate (3-BrPA) has a very similar structure with lactate, which can enter cells on the same carrier monocarboxylic acid transporters (MCTs) as lactate. Once enter into cells, 3-BrPA can inhibit two energy producing systems, including glycolysis and mitochondrial oxidative phosphorylation (Azevedo-Silva et al., 2016). As an alkylating agent, 3-BrPA has many targets, and one of its major targets leading to a metabolic catastrophe is probably hexokinase-2 (HKII), a key enzyme for the conversion of glucose to glucose-6-phosphate in the first step of glycolysis pathway (Chen et al., 2009; Galina, 2014; Ko et al., 2001; Pedersen, 2007; Shoshan, 2012). It has been identified 3-BrPA induces a covalent modification of HKII to inhibit the activity of the enzyme, and dissociates it from the mitochondria, this event promotes the release of the apoptosis inducing factor (AIF) thus triggering apoptosis (Chen et al., 2009). Given the high dependence of tumor cells on glycolysis and the pivotal role in the control of tumorigenesis by glycolysis inhibition, 3-BrPA has been extensively studied in anti-cancer therapy(Mathupala et al., 2009; Shoshan, 2012). Thus, its potential pharmacological functions correlated with antitumor activity has been increasingly discovered in recent decades.
On the other hand, in insects, hexokinase is also a pivotal enzyme that catalyzes the second step in the chitin biosynthesis pathway, which transfers a phosphate group from ATP to β-d-glucose to form glucose-6-phosphate for providing a substrate for the next step of chitin synthesis (Muthukrishnan et al., 2012; Zhu et al., 2016). Whereas, whether interference with glycolytic pathway will affect chitin synthesis is still unknown, and the regulatory mechanism between the glycolysis and chitin biosynthetic pathway remain unclear.
To date, few studies have focused on the effects of 3-BrPA-incuced glycolysis inhibition on the growth and development and chitin synthesis in insects. In this study, to uncover the regulatory relationship between glycolysis and chitin biosynthesis in insects, we investigated the effects of glycolytic inhibitor 3-BrPA on the growth and development, as well as the chitin synthesis in fall webworm, Hyphantria cunea larvae, a notorious defoliator in the forestry. The findings provide a novel insight into the mechanism of glycolytic inhibitor in regulating the growth and development of insects, and lay a foundation for the potential application of glycolytic inhibitors in pest control as well.
Section snippets
Reagents
The 3-BrPA (purity ≥95%) was obtained from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China), and dissolved in DMSO before use. The trehalase content assay kit was purchased from SuZhou Grace Biotechnology Co.,Ltd. (SuZhou, China). The glucose-6-phosphate dehydrogenase (G6PDH) activity assay kit, TRIzol reagent and MightyScript First Strand cDNA Synthesis Master Mix were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). TB Green Premix Ex Taq (Tli RNaseH Plus) and the glycogen
3-BrPA restrained the growth and development of H. cunea larvae
To investigate the effects of 3-BrPA on the growth and development of H. cunea larvae, three doses of 3-BrPA (20, 50, and 100 mg/g) were used for bioassay. The results showed 3-BrPA had a high lethality to H. cunea larvae, and the lethal time was positively correlated to the concentration of 3-BrPA. As shown in Fig. 1A, 100 mg/g 3-BrPA caused 100% corrected mortality at the 7th day, whereas 20 mg/g groups resulted in 100% corrected mortality at the 18th day after treatment. Furthermore, 3-BrPA
Discussion
As an analogue of pyruvate, 3-BrPA was found to be an effective inhibitor of glycolysis, which can inhibit cellular ATP production, increase oxidative stress, and reduce GSH level (Shoshan, 2012). As is known, aerobic glycolysis is closely associated with tumorigenesis and plays important roles in maintaining the malignant behaviors of the cancer cells, thus 3-BrPA acts as a glycolytic inhibitor and was extensively studied in cancer treatment for its remarkable efficacy in preventing tumor
Acknowledgments
This work was supported by the Fundamental Research Fund for the Central Universities (2572020DR09 and 2572018BA04), the Natural Science Foundation of Heilongjiang Province (LH2021C010), and the Jilin Province Science & Technology Development Plan Project (20180201060NY).
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