Obesity is associated with elevated blood glucose levels and insulin resistance which is critical in defining the risk and development of T2D 22. In our study, both excess weight gain increase as a measure of obesity and elevated blood glucose levels as a marker of T2D were detected when human-liked processed food as defined cafeteria diet was applied to the CAF group mothers. Besides, the expression of glucagon was assessed with IHC in the mother groups, since T2D is a state of relative glucagon overproduction (Godoy-Matos A. F., 2014). As a result, higher glucagon expression was observed in the mother CAF group compared to the control group, thus reconfirming the diabetic status of the mother CAF group. On the other hand, it could also be an indicator of an increase in the alpha cell population which may indicate the increased beta cell dedifferentiation in T2D of these subjects.
The average body weight of 20 days old offspring of the control group was higher than that of the CAF group which was in accordance with the findings of the previous study, which reported that offspring of overweight mothers gained less weight and grew less than babies of normal-weight mothers from just after birth to three months 20. Also, we found that the blood insulin levels of 20 days old offspring were greater in the control group compared to the CAF group, suggesting that maternal obesity influences offspring T2D susceptibility, as plasma insulin level is an important indicator for T2D susceptibility 21.
Beta-cell dedifferentiation to endocrine progenitor like cells under hyperglycemia induced metabolic stress conditions was an alternative proposed mechanism for diabetic loss of functional beta cell number and insulin content 23. To date, studies using animal experiments have found that pancreatic cells of mice exhibit a progenitor cell like character and undergo dedifferentiation in response to hyperglycemia 14. According to the gene expression analyses, it was found in our study that progenitor gene expressions such as Neurogenin 3, Chromogranin A, and Vimentin were upregulated in both mother and offspring CAF groups compared to the control group in agreement with previously reported studies(Roefs et al., 2017, Herold et al., 2018). These studies have also demonstrated that pancreatic endocrine cell subtypes, particularly alpha cells, exhibit progenitor cell characteristics in this transformation process19 which may be also an explanatory finding for hyperglucagonemia seen in diabetes patients 14. As a result, the pancreatic hormone gene expressions of insulin and glucagon which showed that the insulin gene expression was lower and the glucagon gene expression was higher in all the CAF groups than the control group, may help to explain our hypothesis.
In a previous study, pancreatic tissue sections of T2D patients showed a 3-fold increase in the number of Chga A-positive endocrine cellsin all ages of offspring in the CAF groups. (Broedbaek & Hilsted, 2016). Since Chga is a pancreatic endocrine progenitor marker like Neurog3, higher expression levels of Chga may suggest that offspring of the CAF group endocrine pancreatic cells may be more progenitor-like than those of the control group. Additionally, it was also shown that mature functional pancreatic beta cell marker gene FoxO1 expression was significantly lower in the CAF groups than in the control groups. FoxO1 as a transcription factor is functioning in beta cell signal integration and regulation 27. Since FoxO1 has been also found to determine cell fate under metabolic stress, there is an inverse relationship between FoxO1 and beta cell differentiation, and FoxO1 is required to maintain beta cell identity 14. Our results indicated that FoxO1 plays a crucial role in the beta cell dedifferentiation process since its expression levels were lower in all control groups vs. the CAF groups. Our results, also revealed that these marker gene expression levels were significantly different in all age groups of offspring demonstrating that maternal obesity has a great effect on offspring’s pancreas as they reach adulthood.
Western blot analyses for insulin protein expression showed that intracellular proinsulin levels in the mother and 90 days old offspring groups were lower in the CAF groups compared to the control groups. However, in the 20-day-old offspring, where the effect of maternal obesity was more clearly seen in the study's earlier findings, there was no difference between the CAF and control groups. The normalization of the plasma insulin levels of the 90-day-old offspring in the CAF group and the fact that insulin gene expression was lower in all mother and offspring CAF groups compared to the control group suggests that mature insulin secretion can occur at a certain level against insulin resistance, which occurs in diabetes and prediabetes. The presence of more intracellular proinsulin in pancreatic tissue samples of the 20 days old offspring CAF group compared to the 90 days old offspring CAF group may be a sign of a defect in the insulin secretion mechanism demonstrated in T2D cases in different previous studies 28. Recent genome-wide association studies have discovered genes related to the processing of proinsulin, and carboxypeptidase E (CPE) play a key role in this prohormone convertase activity (Chen et al., 2022). Studies on the production of insulin reveal that Cpefat/Cpefat mice had much higher levels of the insulin precursor, proinsulin, indicating that a malfunctioning CPE impacts a proinsulin cleavage step that occurs earlier (Cawley et al., 2004). Therefore, a similar defect may have occurred in the intracellular insulin maturation and secretion mechanism of the 20 days old offspring group due to the intense metabolic stress caused by maternal obesity.
Epigenetic modifications presumably have a crucial role in the heritability of metabolic diseases like obesity and T2D. In particular, rodent studies showed that epigenetic mechanisms are responsible for linking maternal nutritional imbalances to the risk for T2D in adulthood (Lecoutre et al., 2021). Therefore, the maternal obesity effect observed in the offspring groups in our study may be mostly attributable to epigenetic changes such as various DNA methylation profiles and chromosome modifications transferred from mother to offspring. To elucidate these inherited epigenetic mechanisms, more comprehensive studies on similar study models are required.