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. 2025 Apr 30;15:15198. doi: 10.1038/s41598-025-99473-z

The mechanism of maternal inheritance of glycolipid metabolism disorder in a zebrafish model of type 2 diabetes

Fuqi Yang 1,#, Ying Zhang 2,#, Panyu Ju 2, Leyu Li 2, Yu Gong 2, Qian Zhang 2, Jiaolong Huang 2, Peng Duan 2,✉, Xingjian Zhou 2,✉
PMCID: PMC12044153  PMID: 40307382

Abstract

In recent years, the number of people with type 2 diabetes mellitus (T2DM) has been increasing, and there is obvious familial aggregation of T2DM. Pregestational and gestational diabetes mellitus are the most common chronic conditions during pregnancy. However, the mechanism by which maternal preconception hyperglycaemia affects glucolipid metabolism in the offspring is not fully understood. Zebrafish have been widely used to construct T2DM models. In this study, we established a successful T2DM model of female zebrafish by immersing them in a 2% glucose solution for 28 days. The results showed that female zebrafish in the T2DM group exhibited damage to the ovaries and livers. Fasting blood glucose, insulin, cholesterol and triglycerides were increased in the T2DM group compared to the control group. Moreover, a delayed hatching rate, increased yolk sac area, body length and heart rate and decreased blood flow velocity were observed in the F1 larvae from the maternal zebrafish with T2DM. Glucose, insulin and lipid metabolism were prominently affected in F1 offspring. Importantly, the influence on fasting blood glucose and insulin could persist into adulthood in F1 offspring of zebrafish with maternal T2DM. Transcriptomic results indicated that the signalling pathways of gluconeogenesis, fat digestion and absorption and cholesterol and amino acid metabolism were enriched and perturbed in F1 larvae from zebrafish with maternal T2DM. This study emphasised the impacts of maternal preconception zebrafish hyperglycaemia on glycolipid metabolism in the offspring, transferring the maternal origin of the disease to the preconception stage and providing a reference for further research on the aetiology of glycolipid metabolism disorders.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-99473-z.

Keywords: T2DM, Maternal exposure, Glycolipid metabolism, Intergenerational effect, Zebrafish

Subject terms: Genetics, Molecular biology, Diseases, Endocrinology, Medical research, Pathogenesis

Introduction

Diabetes mellitus (DM) is a growing global health threat, T2DM in particular has increasingly affected the younger population. In recent years, relative increases in the incidence and prevalence of T2DM have been observed among younger populations, especially in adolescents and children1. T2DM is primarily caused by lifestyle and genetic factors, and there is prominent familial aggregation of T2DM and related phenotypes, including dyslipidaemia and obesity2. Previous studies have revealed that lifestyle factors, such as lack of physical activity, poor diet and urbanisation, are key contributors to T2DM3,4. However, the molecular mechanisms underlying the adverse effects of maternal T2DM on offspring remain largely unknown.

The developmental origins of health and disease (DOHAD) indicate that an adverse environment in early infancy could affect whole-body metabolism and increase the risk of disease later in life5. Pregestational diabetes mellitus (PGDM) and gestational diabetes mellitus are the most common chronic medical conditions affecting pregnancy6,7. Due to the increasing prevalence of risk factors such as obesity and advanced maternal age, PGDM is likely to continue to increase in the obstetric population8. In Canadian First Nations children, those exposed to PGDM had a higher risk of childhood-onset type 2 diabetes (odds ratio = 14.4) compared to children exposed to gestational diabetes mellitus (odds ratio = 4) or those not exposed9. PGDM in mice led to defective insulin secretion in the F1 offspring, even when oocytes were fertilised in vitro and transferred into healthy female mice for development10. These findings suggest that maternal hyperglycaemia before pregnancy has potential long-term effects on foetal health.

It has been reported that PGDM and gestational diabetes mellitus pose additional risks to the embryo, the foetus and the course of the pregnancy11. Many studies have reported on the adverse effects of gestational diabetes mellitus on the offspring of humans12and other mammals, such as pigs13, mice, and rats14. However, because mammalian fertilisation occurs internally and the development of the foetus is affected by the intrauterine environment, few studies have focused on the effects of PGDM on offspring. A previous study using one-cell zygote transplantation found that PGDM led to congenital malformations and growth retardation in offspring15. Another study revealed that maternal hyperglycaemia before pregnancy caused abnormal glucose tolerance in F1 mice by embryo transfer in vitro10. However, the mechanisms by which maternal hyperglycaemia before pregnancy affects offspring development still need further investigation.

Zebrafish embryos are fertilized externally, which can avoid the effects of a hyperglycaemic maternal environment, and the transparency of the embryos facilitates the observation of early development. Recent data demonstrated that zebrafish share the same enzymes and regulatory pathways for glucose metabolism as mice and humans16,17. There are three main methods for constructing a zebrafish T2DM model: glucose immersion, diet induction and gene knockout18. In the study, we established a female zebrafish model of T2DM by glucose immersion, evaluated the effects of maternal preconception hyperglycaemia on glucose and lipid metabolism in the F1 offspring and explored the mechanism by transcriptome sequencing. Our findings support the idea that PGDM causes disorders of glucolipid metabolism in the offspring and provide a basis for further research into its aetiology and treatment.

Results

Biochemical indicators in the F0 adult female zebrafish

Female zebrafish were soaked in 2% glucose for 28 days, the body weight and length were measured and the body mass index (BMI)19, calculated as body weight divided by body length squared (body weight/body length²)20 was calculated (Fig. 1a). The results showed that the BMI and the content of total cholesterol (TC) and triglycerides (TG) were significantly increased in the exposed group compared with the control group (Fig. 1b, c and d). The fasting blood glucose (FBG) and insulin (FINS) levels in the exposed group were significantly elevated on days 14, 21 and 28 of the experiment compared to the control group (Fig. 1e and f). These results showed that disturbances of glucose and lipid metabolism occurred in female zebrafish after glucose exposure. This suggested that a T2DM zebrafish model had been established successfully by 2% glucose immersion.

Fig. 1.

Fig. 1

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The body mass index (BMI), total cholesterol (TC), triglycerides (TG), fasting blood glucose (FBG) and insulin (FINS) in female zebrafish after 2% glucose immersion. (a) Experimental design for building type 2 diabetes mellitus (T2DM) model in female zebrafish. (b) BMI (n = 10 fish per group), (c) TC content (n = 6 biological replicates per group), (d) TG content (n = 6 biological replicates per group), (e) fasting blood glucose (n = 6 biological replicates per group) and (f) insulin content (n = 6 biological replicates per group). Values are presented as mean ± SEM. (T2DM: 2% glucose group). The different letters indicate significant differences among different treatments in the same generation (P < 0.05).

Histopathological alternations in the livers and ovaries of F0 adult zebrafish

Haematoxylin and eosin (H&E) staining was performed on the control and T2DM groups (Fig. 2a). In the control group livers, hepatocytes were tightly and compactly arranged with centrally located spherical nuclei (Fig. 2b). By contrast, in maternal zebrafish of the T2DM group, hepatocytes exhibited an irregular arrangement, and pathological sections showed obvious hepatocyte degeneration and vacuolation throughout the liver tissue (Fig. 2c).

Fig. 2.

Fig. 2

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Histopathological alternations in livers and ovaries in the control and T2DM groups. (a) Experimental design for haematoxylin and eosin (H&E) staining. H&E staining of livers (b) in the control group, (c) in the T2DM group. Scale: 100 μm. Blue arrows indicate steatosis; green arrows indicate inflammatory cell infiltration. H&E staining of ovaries (d) in the control group, (e) in the T2DM group. (f) Percentage of different stages of oocytes in the ovaries (n = 6 fish per group). Values are presented as mean ± SEM. The different letters indicate significant differences among different treatments in the same generation (P < 0.05). Scale: 100 μm. Perinucleolar oocytes (PO), cortical alveolar oocytes (CO), early vitellogenic oocytes (EV) and late vitellogenic oocytes (LV).

Ovarian histology showed a significant increase in the percentage of late vitellogenic oocytes (LV) and a significant decrease in the percentage of primary oocytes (PO) in the maternal zebrafish of the T2DM group compared with those of the control group (Fig. 2d, e and f). The percentage of cortical-alveolar oocytes (CO) and early vitellogenic oocytes (EV) showed no significant difference between the T2DM and control groups (Fig. 2d, e and f). These results show that T2DM causes lipid accumulation in the liver and leads to abnormal oocyte development in female zebrafish.

Assessment of developmental indicators in F1 embryos/larvae

On days 14, 21 and 28 of exposure, treated female zebrafish were mated with untreated male zebrafish (Fig. 3a). The result demonstrated that the total spawning eggs in the T2DM group showed no significant alternations compared with the control group (Fig. 3c). However, at 24 h post fertilisation (hpf), spontaneous movement was significantly inhibited in the F1 embryos from the maternal zebrafish in the T2DM group and reduced in a time-dependent manner with increasing days of glucose immersion (Fig. 3d). The hatching rate of F1 embryos from the maternal zebrafish with T2DM was significantly increased at 60 hpf compared with the control group (Fig. 3b and e). However, the survival rate of F1 larvae from the maternal zebrafish with T2DM was not significantly affected at 72 hpf compared with the control group (Fig. 3f).

Fig. 3.

Fig. 3

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Assessment of the effects on reproductive capacity and development indicators of F1 offspring from maternal zebrafish with T2DM. (a) Experimental design for developmental assessment of the F1 offspring. (b) Representative images of F1 embryos at 60 h post fertilisation (hpf), (c) total spawning eggs of female zebrafish (n = 6 fish per group), (d) frequency of spontaneous movements (n = 15 fish per group), (e) hatching rate (n = 6 wells per group) and (f) survival rate (n = 6 wells per group). Values are presented as mean ± SEM. The different letters indicate significant differences among different treatments in the same generation (P < 0.05).

Compared with the control group, abnormal development was observed in the F1 larvae from the maternal zebrafish with T2DM (Fig. 4a). The yolk sac area (YSE) and heart rate of F1 larvae from the maternal zebrafish with T2DM were significantly increased at 72 hpf compared to the control group (Fig. 4b, c and d). The body length of F1 embryos at 96 hpf was also significantly increased in T2DM group compared with the control group and showed a time-dependent increase with increasing days of glucose immersion (Fig. 4e and f). A significant reduction of the blood flow velocity inF1 larvae from the maternal zebrafish with T2DM was shown at 60 hpf compared to the control group (Fig. 4g). These results indicated that T2DM in maternal zebrafish affected the development of F1 offspring.

Fig. 4.

Fig. 4

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Developmental assessment in F1 offspring from maternal zebrafish with T2DM. (a) Experimental design for the developmental assessment of F1 offspring. (b) Representative images of F1 larvae at 72 hpf (n = 15 fish per group), (c) yolk sac area (n = 15 fish per group), (d) heart rate (n = 15 fish per group), (e) representative images of F1 larvae at 96 hpf, (f) body length (n = 15 fish per group), (g) blood flow velocity (n = 15 fish per group). Values are presented as mean ± SEM. The different letters indicate significant differences among different treatments in the same generation (P < 0.05).

Evaluation of biochemical indicators in F1 offspring

The F1 larvae were collected and stained with Oil Red O (Fig. 5a). The results of whole-body Oil Red O staining exhibited that increased lipid deposition and deeper staining were observed in the yolk sacs of F1 zebrafish larvae from maternal zebrafish with T2DM compared to those from control zebrafish (Fig. 5a and b). The average optical density (AOD) was significantly increased in the F1 larvae from the group with maternal T2DM compared to the control group (Fig. 5c). The levels of TC and TG in the F1 zebrafish larvae from the T2DM group were also significantly enhanced at 72 hpf compared with the control group (Fig. 5d and e). These findings suggested that maternal T2DM in zebrafish affected lipid metabolism in F1 larvae, leading to the accumulation of lipids in the yolk sac.

Fig. 5.

Fig. 5

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Lipid metabolism and glucose metabolism assessment in F1 larvae from maternal zebrafish with T2DM. (a) Overview of experimental design. (b) Images of Oil Red O staining in the control and T2DM groups, (c) the average optical density (AOD) of lipid droplet accumulation (n = 10 fish per group), (d) TC content (n = 6 biological replicates per group), (e) TG content (n = 6 biological replicates per group). The contents of (f) Glucose (GLU) (n = 6 biological replicates per group); (g) insulin (INS) (n = 6 biological replicates per group) in the F1 larvae at 72 hpf, (h) fasting blood glucose (n = 6 biological replicates per group) and (i) insulin content (n = 6 biological replicates per group) in F1 adult zebrafish from the 28 day exposure group. Values are presented as mean ± SEM. The different letters indicate significant differences among different treatments in the same generation (P < 0.05).

Glucose levels (Glu) were significantly reduced in the F1 zebrafish larvae from the group with maternal T2DM (Fig. 5a and f), while insulin levels (INS) were significantly increased in the F1 larvae from the zebrafish with maternal T2DM compared to the control group and showed a time-dependent increase with increasing days of glucose exposure (Fig. 5g). Moreover, we compared the fasting blood glucose and insulin levels in F1 adults from the 28-day exposure group and found that the levels of fasting blood glucose and insulin in F1 adults from zebrafish with maternal T2DM were significantly increased compared to the control group. (Figure 5h and i). These results indicate that disordered glucose metabolism and insulin resistance in the F1 offspring of zebrafish with maternal T2DM could persist into adulthood.

Behaviour assays in the F1 zebrafish larvae

The locomotor activity of F1 zebrafish larvae at 120 hpf was analysed. The results showed that although the trajectories differed between the T2DM and control groups, the total distance travelled showed no significant differences in F1 larvae from the maternal zebrafish with T2DM compared with the control group (Fig. S1a, 1b, 1c). However, the current study demonstrated that the active frequency of the F1 larvae from the maternal zebrafish with T2DM was significantly inhibited compared to the control group (Fig. S1d). The results demonstrated that maternal zebrafish with T2DM affected locomotor activity in F1 larvae.

Transcriptomic sequencing analysis

To explore the mechanism of glucose and lipid metabolism disorder in the F1 larvae from zebrafish with maternal T2DM, transcriptomic analysis was performed. Genes with fold change ≥ 1 and P < 0.05 were identified as differentially expressed genes (DEGs). The significance of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was mapped to interpret the biological implications of DEGs. P < 0.05 was considered the threshold for significance for GO terms and KEGG pathway enrichment. The results exhibited that a total of 167 differentially expressed genes (DEGs), including 57 up-regulated and 110 down-regulated DEGs, were found in F1 larvae from maternal zebrafish with T2DM compared with the control group (Fig. 6a).

Fig. 6.

Fig. 6

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Transcriptome analysis of F1 larvae at 96 hpf from maternal zebrafish with T2DM. (a) Volcano Plot of differentially expressed genes (DEGs). Upregulated genes shown in red, downregulated genes in green. (b) Gene Ontology (GO) Enrichment Analysis of DEGs. Biological processes shown in red, cellular components shown in blue, molecular functions shown in green. (c) Kyoto Encyclopedia of Genes and Genomes (KEGG)61 enrichment analysis of DEGs. The highlighted signalling pathways are related to lipid and glucose metabolism. (d) Protein-protein interactive (PPIN) of DEGs. The size and colour intensity represent the degree (number of interactions), n = 4 biological replicates per group.

In the GO enrichment analysis, biological processes (BP) principally included phenomena such as xenobiotic metabolic processes, tryptophan catabolic processes and endocrine pancreas development. The molecular functions were predominantly fatty acid binding, steroid hydroxylase activity and lipopolysaccharide binding (Fig. 6b). In the KEGG enrichment analysis, the pathways primarily enriched were linoleic acid metabolism, phenylalanine metabolism, fat digestion and absorption, cholesterol metabolism and peroxisome proliferator-activated receptor (PPAR) signalling pathways (Fig. 6c). These results suggested that maternal zebrafish with T2DM induced dysregulation of glycolipid metabolism in F1 larvae.

Furthermore, protein-protein interactive (PPIN) analysis was performed on the DEGs (Fig. 6d). The results indicated that the genes fga, cox7c, rps25, rps28 and rps29, which were both hub genes and glycolipid metabolism-related genes, affected in the F1 larvae from maternal zebrafish with T2DM.

Validation of gene expression by qRT-PCR

To validate the results of RNA-Seq, qRT-PCR was performed and demonstrated that the expression of genes (g6pca2, apoc1, afp4, fabp2 and apoa4b3) related to glucose and lipid metabolism was significantly decreased in the F1 larvae from the maternal zebrafish with T2DM compared to the control group (Fig. 7a and b). Additionally, the transcription of genes (gsto2, ddc and idh1) involved in amino acid metabolism was significantly reduced in the F1 larvae from the maternal zebrafish with T2DM compared to the control group (Fig. 7c and d). In our study, the correlation analysis showed that the results of qRT-PCR were consistent with the RNA-Seq (Fig. S2).

Fig. 7.

Fig. 7

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The mRNA expression of genes related to glucose and lipid metabolism in F1 larvae. (a) Heat map of mRNA expression (n = 4 biological replicates per group) and (b) the results of qRT-PCR for key genes in signalling pathways related to glucose and lipid metabolism (n = 6 biological replicates per group). (c) Heat map of mRNA expression (n = 4 biological replicates per group) and (d) the results of qRT-PCR for key genes in signalling pathways related to amino acid metabolism (n = 6 biological replicates per group). Values are presented as mean ± SEM. The different letters indicate significant differences among different treatments in the same generation (P < 0.05).

Discussion

As the number of young people with T2DM increases, so does the prevalence of PGDM compared with the last 20 years11. Maternal exposure to adverse conditions early in life or during pregnancy greatly impacts the offspring and may lead to lasting intergenerational genetic effects21. In the current study, we successfully established a model of T2DM in female zebrafish by immersing them in 2% glucose. The results showed that maternal T2DM in zebrafish resulted in liver and ovarian damage. Moreover, maternal hyperglycaemia not only induced developmental abnormalities in F1 offspring but also led to glycolipid metabolism disorders in F1 larvae and adult zebrafish. Transcriptomics suggested that the mechanisms involved were possibly related to key signalling pathways that regulate glycolipid metabolism. These findings indicated that maternal hyperglycaemia before pregnancy leads to abnormal development and persistent glycolipid metabolic disorders in F1 offspring.

Previous studies have shown that unhealthy diet and lifestyle habits adversely affect the ovarian environment and further affect oocyte quality and early embryo development22,23. We found that maternal T2DM in zebrafish increased late vitellogenic oocytes (LV) in the ovaries, which may be associated with the observed trend of increased egg production in female zebrafish, although no statistically significant differences were observed. A previous study showed that during oogenesis, maternal diabetes in mice led to abnormal development in the F1 offspring24, as well as glucose intolerance and insulin resistance25. Epidemiological studies have shown that PGDM is closely associated with an increased risk of foetal complications, including preterm birth, large for gestational age infants, macrosomia and neonatal hypoglycaemia26,27. Consistent with our results, we observed that maternal hyperglycaemia before pregnancy caused developmental malformations, hypoglycaemia and insulin resistance (IR) in F1 offspring. Furthermore, in our study we observed decreased blood flow but increased heart rate in F1 larvae from the maternal zebrafish T2DM group. A clinical study in the Danish National Birth Cohort found that offspring of mothers with hyperglycaemia during pregnancy had increased heart rate, higher blood pressure, cardiac function disturbances, and an adverse lipid profile, all linked to insulin resistance28,29. A rat model study showed that PGDM offspring had cardiac dysfunction with reduced ejection fraction and fractional shortening, driven by hyperinsulinemia30. Clinical studies have confirmed that maternal hyperglycaemia also impairs neonatal cardiac function, with insulin resistance and inflammation damaging microvessels31, reducing blood flow velocity32. The increased heart rate may compensate for the decline in cardiac function and blood flow velocity. These findings could explain the increased heart rate and reduced blood flow velocity observed in F1 larvae. The results suggested the important impacts of maternal hyperglycaemia before pregnancy on the health of the offspring.

It has been shown that when female mice were fed a high-fat diet prior to pregnancy, their offspring developed obesity, abnormal glucose tolerance and insulin resistance in adulthood25. Clinical research confirmed that neonatal hyperinsulinemia at birth is a primary cause of neonatal hypoglycaemia33. The present study found that maternal zebrafish with T2DM result in hypoglycaemia and IR in the F1 offspring. A recent study reported that immersing adult zebrafish in a high-glucose environment for 60 days significantly increased the level of glucose, TG and TC and induced IR34. A clinical multicentre cohort study indicated that IR may precede obesity, visceral adiposity, and hyperlipidemia, suggesting that IR serve as the central factor linking each component of metabolic syndrome. Changes in glucose and lipid metabolism inevitably occur along with the changes in IR, leading to excessive lipid accumulation35. Another study showed that female mice with IR could cause higher lipid levels with fasting hyperinsulinemia and fasting hyperglycaemia in the offspring36. Therefore, in this study, the IR in the F1 offspring from the maternal zebrafish with T2DM was possibly associated with lipid accumulation. However, whether altered lipid metabolism is a result of hyperinsulinemia and insulin resistance or, conversely, lipid accumulation contributes to the development of insulin resistance, remains to be further investigated.

Glycolipid metabolism plays an essential role in lipid regulation and is closely associated with the development of T2DM37. Our study found that DEGs were mainly enriched in the pathways related to glycolipid and amino acid metabolism in the F1 larvae from the maternal zebrafish T2DM group. These pathways are interconnected and collectively regulate glucose and lipid metabolism in zebrafish. Research on mice with gestational diabetes showed that the PPAR pathway was upregulated simultaneously with linoleic acid, cholesterol metabolism and fat digestion38. The PPAR signalling pathway is a master regulator of lipid, cholesterol and carbohydrate metabolism39; it can be activated by fatty acids and their derivatives from linoleic acid metabolism and plays a key role in energy storage, fatty acid oxidation and insulin sensitivity40. Additionally, changes in glucolipid metabolism significantly impact the metabolism of phenylalanine, tyrosine and tryptophan41,42. Therefore, the alteration of these signalling pathways, such as the PPAR signalling pathway, fatty acid metabolism and cholesterol metabolism caused by T2DM of maternal zebrafish possibly led to the disturbance of glycolipid metabolism in F1 offspring.

G6pca.2is a key regulator of glucose homeostasis43and plays an important role in gluconeogenesis44. In the present study, significant down-regulation of g6pca.2 may be possible with decreased glucose levels. Several genes related to lipid metabolic processes (fabp2, fabp1b.1, afp4, apoc1, apoa4b.3 and cyp3c4) were also reduced in F1 larvae from zebrafish with maternal T2DM. Fatty acid-binding proteins (FABPS) such as fabp2 and fabp1b.1play a crucial role in lipid transport. They bind excess fatty acids to maintain a stable pool within the epithelium25,45and participate in the regulation of lipid sensing and response mechanisms in tissues and cells by transporting lipids to nuclear receptors, such as peroxisome proliferator-activated receptors46. The role of apoc1 in glycolipid metabolism is still under investigation, but studies have confirmed that apoc1is significantly downregulated in children with type 1 diabetes47and may lead to increased high-density lipoprotein (HDL) breakdown and enhanced very low-density lipoprotein (VLDL) production48. Cytochrome P450 s (CYPs) are essential for the metabolism and biotransformation of various xenobiotic compounds. CYPs particularly catalyse the oxidative metabolism of lipophilic compounds, including both exogenous and endogenous organic substances, such as sterols, fatty acids and hormones49,50. Moreover, significant downregulation of several genes, including gsto2, kynu, ddc and idh1, involved in amino acid metabolism, were observed. The genes are associated with tryptophan metabolism, tyrosine metabolism and glutathione metabolism. Amino acids, as key substrates for gluconeogenesis, are closely linked to glucose metabolism and can influence insulin and glucagon secretion50. Hence, we suggest that the downregulation of these glucolipid metabolism-related genes may lead to a decrease in lipid transport and breakdown, consequently affecting lipid accumulation in the F1 larvae of F0 zebrafish with T2DM.

Adverse environmental factors in early life could lead to disease in adulthood51. The present study showed that maternal T2DM in zebrafish induced liver and ovarian damage in F0 zebrafish and led to abnormal development and disorders of glucose and lipid metabolism in F1 offspring. Because it is difficult to rule out the influence of the intrauterine environment, knowledge of the developmental impacts of preconception diabetes on the offspring and the molecular mechanisms is still lacking in clinical practice26,52. Although the reproductive mode is external fertilization, evolutionary conservation of pancreas insulin-sensitive target tissues (muscle and liver) and key mechanisms associated with glucose metabolism show remarkable similarities between zebrafish and certain mammals53, these results provide a theoretical basis for the origin of the disease. However, our study has limitations, whether the effects on glucose and lipid metabolism have transgenerational impacts needs to be investigated and the mechanisms of female germline-dependent epigenetic inheritance remain largely unclear.

Conclusion

In conclusion, our study found that maternal T2DM induced liver and ovarian damage in F0 zebrafish and disrupted glucose and lipid metabolism in F1 offspring. Furthermore, we found that the effects on glycolipid and insulin could persist into adulthood. The transcriptomic results indicated that cholesterol, fatty acid and amino acid metabolism were primarily affected in F1 zebrafish larvae from zebrafish with maternal T2DM. These findings provide more evidence for the early life origins of disease, transferring the maternal origin of disease in offspring to the preconception stage. Therefore, it is necessary to enhance preconception screening for DM and improve the gamete environment to mitigate maternal effects on glucolipid metabolism in offspring. Further studies are needed to determine whether the effects of maternal T2DM on glycolipid metabolism in offspring have transgenerational effects and to elucidate the underlying epigenetic mechanisms.

Materials and methods

Zebrafish maintenance

Adult AB strain zebrafish (5 months old) were procured from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The zebrafish were maintained in a recirculation system designed to house zebrafish with the water temperature at 26–28 ℃, pH 7.0–7.2 and a 14 h/10 h light/dark cycle. Adult zebrafish were fed with newly hatched brine shrimp twice a day.

Zebrafish embryos obtained through female and male mating were cultured in ultrapure water at a constant temperature of 28 ℃.

Establishment of a T2DM model in female zebrafish

Glucose (CAS 50–99-7, 99.7% purity) was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Hyperglycaemia in zebrafish was induced by administering a high-sugar diet. AB strain adult female zebrafish (5 months) were randomly selected and divided into two groups. Zebrafish in the experimental group were immersed in 2% glucose solution to establish a T2DM model; zebrafish in the control group were raised in standard system water. Both groups were maintained at a density of 30 zebrafish per 8 L of water (three tanks per group), with the water was refreshed once a day and were fed the same volume of brine shrimp twice daily. The whole experiment was performed over 28 days. Based on the latest research, studies have utilized glucose exposure for over 30 days to induce diabetic phenotypes in zebrafish to better simulate the long-term development of human type 2 diabetes34. In our study, pre-experimental results suggested that a continuous 28-day exposure period produced a more stable model compared to a 14-day exposure or an intermittent 28-day exposure. With slight modifications to previous methods, we adopted this continuous 28-day exposure to establish a T2DM model in female zebrafish.

Measurement of biochemical parameters in F0 female zebrafish

At the end of the experiment, the adult female zebrafish were subjected to a 12-hour fasting period before sampling. Zebrafish were anaesthetised with 0.03% tricaine (MS-222). The body weight and length were measured, and BMI was calculated as body weight/body length2 (n= 10 per fish group). The method for blood sampling and biochemical analysis was adapted from a previous study54. The blood was taken from the caudal vein using capillaries and centrifuged at 26 ℃ and 3000 rpm for 5 min. The GLU assay kit (A154-1-1), TC assay kit (A111-1-1), and TG assay kit (A110-1-1) were obtained from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). For each parameter, 12 fish were used, with serum from 2 fish pooled into one sample, resulting in 6 biological replicates (n = 6 biological replicates per group). A 2.5 µL volume of supernatant from each sample was used for measurement. Glucose content was determined using the glucose oxidase (GOD) method, while TC and TG levels were measured using the GPO-PAP method. The INS assay kit (m1838323) was obtained from Shanghai MLBIO Biotechnology (Shanghai, China). A total of 24 fish were used, with serum from 4 fish pooled into one sample, resulting in 6 biological replicates (n = 6 biological replicates per group). For each measurement, 10 µL of supernatant from each sample was used and diluted to 50 µL, following the instructions, before analysis. Insulin levels were measured using the enzyme-linked immunosorbent assay (ELISA) method.

Histopathological analysis

After being soaked in 2% glucose for 28 days, fresh ovary and liver tissues (n= 6 fish per group) from F0 adult zebrafish in each group were fixed with 4% paraformaldehyde (PFA). The samples were dehydrated in ethanol and xylene and embedded in paraffin. Embedded tissues were cut into 5-µm-thick sections and stained with H&E55. Histological sections of ovaries and livers were selected randomly and scanned by a digital slide scanner (Pannoramic 250, 3D Histech, Hungary). The number of cells at different stages in the ovaries was counted utilising a light microscope (Olympus CX31).

Development assessment of F1 embryo-larvae

On days 14, 21, and 28 of the experiment, treated female zebrafish (n= 6 fish per group) were randomly selected and paired with untreated male zebrafish (1:1). Each pair was individually transferred to a spawning aquarium filled with purified system water and placed in spawning boxes separated by isolation boards overnight. The following morning, the lights were turned on, and the isolation plates were removed to facilitate natural mating. Fertilized eggs were collected within 30 min of spawning, and the number of spawned eggs per female was recorded55. Then, the fertilized embryos within 2 hpf were placed in a 6-well plate containing 5 mL ultrapure water, with 50 eggs per well, and the water was renewed once a day. The survival, deformity and hatching rates (n = 6 wells per group) were recorded every 12 h, and the frequency of spontaneous coiling (24 hpf), yolk sac area (72 hpf) and body length (96 hpf) of F1 offspring (n = 15 fish per group) were observed under a stereomicroscope (SZX7, Olympus, Antwerp, Belgium). The blood flow velocity (60 hpf) and heart rate (72 hpf) of F1 zebrafish larvae (n = 15 fish per group) were measured using MicroZebraLab software (ViewPoint, France).

Staining with oil red O and measurement of TG and TC in F1 larvae

F1 embryos were raised in ultrapure water to 72 hpf and then anaesthetised and fixed in 4% PFA for 12 h at 4 °C (n= 10 fish per group). Oil Red O staining was performed according to a previous study56. Briefly, the F1 zebrafish larvae were fixed and washed three times with phosphate-buffered saline (PBS) and subsequently incubated in 60% isopropanol for 30 min and stained with fresh 0.3% Oil Red O for 3 h. After that, the larvae were washed three times with 60% isopropanol and prepared for microscopic observation. The larvae were observed and photographed under a light microscope (Olympus, CX31, Japan). Quantitative analysis was performed using ImageJ software (Rockville, MD, USA).

To measure the content of TG, TC, GLU, and INS in F1 larvae, preliminary experiments showed that a sample size of 100 larvae resulted in the most stable linear relationship in the standard curve. Therefore, each sample consisted of 100 larvae, with six samples measured per group (n= 6 biological replicates). Larvae were collected at 72 hpf. The tissues were homogenized with PBS at a ratio of 1:9 (weight/volume) and centrifuged at 4 °C and 2500 rpm for 15 min. The supernatant was then collected for subsequent analysis. The protocols were conducted using previously published methods34. A 2.5 µL volume of supernatant from each sample was used to measure the content of TG, TC, and GLU, while a 50 µL volume of supernatant from each sample was used to measure the content of insulin. The assay kits and measurement methods are as described above. Additionally, the F1 zebrafish larvae were randomly selected from the T2DM and control groups and raised in clean water. After four months, the blood glucose and insulin levels in the F1 adult zebrafish were measured (n = 6 biological replicates). The assay kits, blood sampling method, and measurement techniques are as described above.

Larval locomotor activity

The behaviour of F1 larvae was also analysed. Zebrafish larvae (n= 12 fish per group) at 120 hpf were selected randomly from the control and T2DM groups and placed into a 48-well plate with one larva per well. Then, they were entered into the behavioural analysis system of zebrafish (ViewPoint, France) to evaluate the locomotor activity after acclimatisation for 10 min at 28 ℃55. To assess swimming behaviour, movements of individual zebrafish larvae in each well over a 10-min period were captured, and the active frequency and average speed of each larva in each group were calculated under the MicroZebraLab software (ViewPoint, France).

RNA sequencing (RNA-Seq) analysis

To further assess the maternal effects of T2DM on the development of F1 offspring, 50 zebrafish larvae at 96 hpf (n= 4 biological replicates per group) were collected for RNA sequencing. 50 zebrafish larvae were collected and placed in cen-trifuge tubes and centrifuged to separate the supernatant as much as possible. The samples were then transported on dry ice to Wuhan Huada Gene Technology Service Co., Ltd. for high-throughput screening, RNA extraction and purification. Each experimental condition was replicated in four independent samples to ensure the robustness and reliability of the data collected57. Tissue samples were collected and homogenised in 1.5 mL of TRIzol reagent (Beyotime, China), incubated for 5 min, and then centrifuged at 12,000 × g for 5 min at 4 °C. Chloroform (24:1) was added, followed by shaking and centrifugation for 8 min. The upper aqueous phase was transferred, mixed with isopropanol, and incubated at −20 °C for 2 h. After centrifugation at 12,000 × g for 25 min, the pellet was washed with 75% ethanol and centrifuged again for 3 min. The pellet was then air-dried, dissolved in DEPC-treated water, and enriched for mRNA using oligo(dT) beads. The mRNA was fragmented, reverse-transcribed into cDNA, and amplified by PCR. The resulting DNA was circularised and sequenced using a nanofluidic chip. The raw data were filtered with SOAPnuke (v1.5.6), aligned with HISAT2 (v2.1.0) and Bowtie2 (v2.3.4.3)58, quantified with RSEM ((v1.3.1)59, and DEGs were identified with DESeq2 (v1.4.5)60. In this study, RNA-Seq data alignment and gene expression analysis were conducted using the Danio rerio (zebrafish) reference genome GRCz11 (NCBI GCF_000002035.6_GRCz11). The reference genome and annotation files, including the transcriptome annotation (Danio_rerio.GRCz11.108.gtf) and primary assembly FASTA sequence (Danio_rerio.GRCz11.dna.primary_assembly.fa), were obtained from NCBI (GCF_000002035.6_GRCz11) and Ensembl (Ensembl GRCz11). The corresponding gene annotation file (GTF) was used to ensure accurate gene expression quantification.

RNA analysis was performed on the Dr. Tom platform. Genes with fold change ≥ 1 and P < 0.05 were identified as DEGs. GO and KEGG were mapped to analyse the biological meaning of DEGs. P < 0.05 was considered the threshold for significance for GO terms and KEGG pathway enrichment. The protein-protein interaction network (PPIN) was analysed in the STRING database (https://string-db.org/) and constructed using Cytoscape software.

Quantitative real-time PCR (qRT-PCR) analysis

Total RNA was extracted from 30 F1 larvae at 96 hpf (n = 6 biological replicates per group) using 1 ml TRIzol reagent (Beyotime, China). RNA purity and concentration were assessed via absorbance measurements, with A260/A280 values between 1.8 and 2.2 considered acceptable. Reverse transcription was performed using the Evo M-MLV RT Reaction Mix for qPCR (+ gDNA wiper) (AG11706, Accutare Biotech, Changsha, China), following the manufacturer’s protocol. Briefly, 1 µg of total RNA was reverse transcribed at 37 °C for 15 min, followed by enzyme inactivation at 85 °C for 5 s.

Real-time quantitative PCR (qRT-PCR) was conducted using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech). The reaction volume was 10 µL, containing 5 µL of SYBR Green Master Mix, 0.2 µL of each primer, 1 µL of cDNA template, and 3.6 µL of nuclease-free water. The qPCR program consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. A melting curve analysis was performed to assess amplicon specificity. DEGs in key differentially expressed pathways were selected for qRT-PCR validation. The primers for each gene were designed by applying the NCBI primer design tool (http://www.ncbi.nlm.nih.gov/tools/primer) and listed in the supplementary information Table S1. The β-actin gene was used as an internal control, and relative gene expression was calculated using the 2−△△Ct method.

Statistical analysis

The data were expressed as mean ± standard error of the mean (SEM). The homogeneity of variances and normality of the data were checked with Levene’s test and the Kolmogorov-Smirnov test using SPSS Statistics Version 25.0 (IBM Corp., Armonk, NY, USA). Tukey’s post hoc test analysis was used to compare the differences between the experimental and control groups, and P < 0.05 was considered the threshold for statistical significance. The graphs were generated using GraphPad Prism 8.0 software (California, USA).

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (3MB, docx)

Acknowledgements

This work, in part, was supported by grants from the Natural Science Foundation of Hubei Province (2022 CFD010, 2023 AFD043, and 2024 AFD035), Beijing Medical Award Foundation (YXJL-2023-0314-0022).

Author contributions

P.D., F.Q.Y. and X.J.Z. designed the experiments. All authors conducted experiments and analyzed data. F.Q.Y. and Y.Z. wrote the first draft of the manuscript and prepared the figures. All authors edited the manuscript.

Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files). The raw RNA-Seq data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession PRJNA1240620 and are publicly accessible at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1240620.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

Zebrafish research in this study was carried out in accordance with ARRIVE guidelines. All methods were in compliant with relevant guidelines and regulations and were approved by the Committee for Animal Experimentation of the Xiangyang No. 1 People’s Hospital, Hubei University of Medicine (No. XYYYE20240149).

Consent for publication

All authors have agreed on the contents of the manuscript.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Fuqi Yang and Ying Zhang contributed equally.

Contributor Information

Peng Duan, Email: meduanpeng@163.com.

Xingjian Zhou, Email: xin.xin1018@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (3MB, docx)

Data Availability Statement

All data generated or analysed during this study are included in this published article (and its Supplementary Information files). The raw RNA-Seq data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession PRJNA1240620 and are publicly accessible at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1240620.

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