3.1 High glucose exposure resulted in a low survival rate of zebrafish larvae
Usually, prolonged high blood sugar induced by the intake of too much sugar takes a toll on the body of both human beings and animals, and hyperglycaemia-induced sugar toxicity and osmotic stress often occur simultaneously(22, 23). Based on this information, to explore the effects of glucose toxicity and minimize the interference of osmotic stress, a suitable concentration of glucose treatment needs to be verified. On the one hand, fertilized eggs were incubated in embryo culture medium containing 0% (control), 0.5%, 1%, 1.5%, 2% and 2.5% glucose and mannitol to explore the influence on the hatchability of zebrafish larvae on the fifth day, and the results showed that there was almost no influence on the hatchability of zebrafish larvae treated with both glucose and mannitol in the concentration range from 0–2.5% (Fig. 1a, b). On the other hand, survival rates were counted for zebrafish larvae that were raised in solutions as described above, and the results suggested that high glucose exposure resulted in a significant decrease in the survival rate at concentrations ranging from 0.5–2.5% (Fig. 1c), while mannitol exposure led to a significant decrease in the survival rate at concentrations ranging from 1.0–2.5% (Fig. 1d). Therefore, the concentration of glucose exposure in this work was determined to be 1.0% solution.
3.2 Hyperglycaemia induced by glucose exposure caused spine malformation and organ oedema in zebrafish larvae
To determine the blood glucose level, the blood glucose of zebrafish larvae was detected at 2 hours and 12 hours after a meal. The results showed that the blood glucose content in zebrafish larvae exposed to glucose was nearly three times as high as that in the control at both 2 hours and 12 hours after a meal (Fig. 2a, b).
Because the toxic symptoms of spinal curvature and oedema of tissues or organs induced by glucose exposure resulted in the death of zebrafish larvae, the effects of glucose exposure on zebrafish larvae were analysed on the tenth day, and the result showed that the activity of almost all larvae was suppressed by glucose exposure. Furthermore, compared with the control group (Fig. 2c), the majority of them appeared to have symptoms of spinal curvature (Fig. 2e, g, i), oedema of tissues or organs (Fig. 2k, h) or both (Fig. 2d, f, j, l).
3.3 Most of the DEGs were related to the apoptosis pathway in the transcriptomic analysis
Mapped data were obtained from clean data filtered using DESeq2 software with the parameters FDR = 0.05 and FC = 2 on a BMK cloud net. On the basis of RNA-seq, a total of 253 differential genes were detected, 187 of which were downregulated and 66 of which were upregulated. According to the above data, a thermogram of differential gene expression was generated, which was related to the zebrafish larval response to sugar toxicity (Fig. 3a).
Combining the significant KEGG pathways and the number of DEGs, the peroxisome, carbon metabolism, apoptosis and phototransduction signalling pathways were suggested to be some of the most significantly enriched pathways, which were usually activated in animals with hyperglycaemia. Compared with the genome-wide background, the DEGs with significant enrichment of more than 5% were selected, and they were related to the peroxisome pathway (5.88%), carbon metabolism pathway (7.06%), phototransduction pathway (7.06%) and the largest number of apoptosis pathways (10. 59%) (Fig. 3b).
3.4 Glucose exposure activated the ROS-accumulation-induced apoptosis in zebrafish larvae in vivo
According to the RNA-seq data, the apoptosis pathway of DEGs needs to be explored preferentially. Under stress conditions, the structure and function of mitochondria, where ROS are primarily produced, need to evaluated to determine whether they are normal. First, ROS accumulation was detected with a DCFH-DA fluorescent probe, and the results showed that glucose exposure increased the accumulation of ROS concentrated in visceral areas much more than that in the control group (Fig. 4a, b). Furthermore, the function of mitochondria was checked using transgenic zebrafish larvae (cms Tg/+ AB), whose mitochondria with normal function glowed bright green under a fluorescence microscope but were faint green when dysfunctional. The results showed that glucose exposure significantly impaired the function of mitochondria in zebrafish larvae compared to the control (Fig. 4c, d). Finally, apoptotic cells were stained with AO dye in vivo, and more apoptotic cells were located in the head and abdomen of zebrafish larvae treated with glucose than in the control larvae (Fig. 4e, f).
3.5 High blood glucose significantly reduced intracellular Ca2+ and increased apoptosis
Because Ca2+ signalling plays an important role in the processes of both apoptosis and autophagy(24), the intracellular Ca2+ content was detected, and it was found that the intracellular free Ca2+ content was significantly lower in glucose-treated zebrafish larvae than in control zebrafish larvae (Fig. 5a, c). To explore the intracellular redox state in zebrafish larvae, intracellular ROS accumulation was measured, and glucose exposure observably improved the ROS content compared to the control (Fig. 5b, f). Based on the detection of apoptosis in vivo, apoptotic cells were measured using FCM, and the results showed that there were many more cells in the early stage of apoptosis in glucose-treated zebrafish larvae than in the control, and although there were still more cells in the late stage of apoptosis in zebrafish larvae exposed to glucose than in the control larvae, the number of late apoptotic cells was relatively low (Fig. 5d, e, g, h). Therefore, in this phase, although a large number of cells had undergone the process of apoptosis, most of them were at the early stage in glucose-treated zebrafish larvae (Fig. g, h, i).
3.6 The antioxidant system was weakened by glucose exposure in zebrafish larvae
The activities of key enzymes of the antioxidant system, which scavenge excess ROS in normal cells, were detected in zebrafish larvae after glucose exposure. Compared with the control, the SOD activity showed a significant decline in glucose-treated zebrafish larvae (Fig. 6a), while the CAT activity was significantly improved (Fig. 6b). To analyse the levels of lipid peroxidation in cells, the MDA content was tested, and the results suggested that glucose exposure caused a significant elevation of MDA in zebrafish larvae compared to the control (Fig. 6c).
3.7 Effects of glucose exposure on the apoptosis of zebrafish larvae
The activity of caspase 3, as a key enzyme of the apoptosis pathway, was measured, and the results showed that glucose exposure significantly improved the activity of caspase 3 in zebrafish larvae (Fig. 7a). Mitochondria are recognized as the initiator of apoptosis, so the mitochondrial membrane potential was tested, and the results suggested that glucose exposure caused prominent depolarization of mitochondrial membrane potential (Fig. 7b).