Prenatal dexamethasone exposure induced pancreatic ß-cell dysfunction and glucose intolerance of male offspring rats: Role of the epigenetic repression of ACE2.

The prevalence of diabetes in children and adolescents has been rising gradually, which is relevant to adverse environment during development, especially prepartum. We aimed to explore the effects of prenatal dexamethasone exposure (PDE) on ß-cell function and glucose homeostasis in juvenile offspring rats. Pregnant Wistar rats were subcutaneously administered with dexamethasone [0.1, 0.2, 0.4mg/(kg.d)] from gestational day 9 to 20. PDE impaired glucose tolerance in the male offspring rather than the females. In male offspring, PDE impaired the development and function of ß-cells, accompanied with lower H3K9ac, H3K14ac and H3K27ac levels in the promoter region of angiotensin-converting enzyme 2 (ACE2) as well as suppressed ACE2 expression. Meanwhile, PDE increased expression of glucocorticoid receptor (GR) and histone deacetylase 3 (HDAC3) in fetal pancreas. Dexamethasone also inhibited ACE2 expression and insulin production in vitro. Recombinant expression of ACE2 restored insulin production inhibited by dexamethasone. In addition, dexamethasone activated GR and HDAC3, increased protein interaction of GR with HDAC3, and promoted the binding of GR-HDAC3 complex to ACE2 promoter region. Both RU486 and TSA abolished dexamethasone-induced decline of histone acetylation and ACE2 expression. In summary, suppression of ACE2 is involved in PDE induced ß-cell dysfunction and glucose intolerance in juvenile male offspring rats.

KDM3A inhibition attenuates high concentration insulin­induced vascular smooth muscle cell injury by suppressing MAPK/NF­κB pathways.

Previous studies have indicated that lysine (K)­specific demethylase 3A (KDM3A) is associated with diverse diabetes­associated cardiovascular complications in response to high glucose levels. However, the effects of KDM3A on the pathological progression of cardiovascular injuries in response to high insulin levels remain unknown. The present study aimed to explore whether KDM3A knockdown may attenuate high insulin­induced vascular smooth muscle cell (VSMC) dysfunction, and to further investigate the underlying mechanisms. Primary VSMCs were isolated from the thoracic aorta of Sprague­Dawley rats. Lentiviral vectors encoding control­small interfering (si)RNA or KDM3A­siRNA were transduced into VSMCs for 72 h, and cells were subsequently incubated in medium containing 100 nM insulin for a further 5 days. Cellular proli-feration, migration and apoptosis were measured by Cell Counting kit­8, Transwell chamber assay and flow cytometry, respectively. Reactive oxygen species (ROS) were detected using the dihydroethidium fluorescent probe. The mRNA expression levels of interleukin­6 and monocyte chemotactic protein­1 were measured by reverse transcription­quantitative polymerase chain reaction. Furthermore, the protein expression levels of KDM3A, mitogen­activated protein kinases (MAPKs), nuclear factor (NF)­κB/p65, B­cell lymphoma 2 (Bcl­2)­associated X protein and Bcl­2 were evaluated by west-ern blotting. Lentivirus transduction with KDM3A­siRNA markedly reduced the elevated expression of KDM3A induced by high insulin stimulation in VSMCs. In addition, inhibition of KDM3A significantly ameliorated insulin­induced VSMC proliferation and migration, which was accompanied by decreased ROS levels, cell apoptosis and inflammatory cytokine levels. Furthermore, KDM3A gene silencing mitigated phosphorylation of MAPKs and NF­κB/p65 activation. In conclusion, KDM3A inhibition may exert numerous protective effects on high insulin­stimulated VSMCs, and the underlying mechanisms may be partly associated with inactivation of MAPK/NF­κB signaling pathways.

Regulatory Forum Opinion Piece: Review-Toxicological Pathology Profile and Regulatory Expectations for Nonclinical Development of Insulins and Insulin Analogues.

The toxicological profile of insulins is exclusively due to exaggerated pharmacology resulting in hypoglycemic findings. Insulin analogues displaying modifications and aimed at improving pharmacokinetics do not induce different toxicity. The main target is the brain displaying neuronal necrosis. Wallerian degeneration of nerves occurs rarely after severe hypoglycemia. These findings are of potential human relevance; nevertheless, these changes are induced in normoglycemic animals whereas diabetic patients suffer from hyperglycemia. Therefore, it is usually not difficult to achieve a therapeutic window for subsequent use in patients. Based upon this and in the absence of classical toxicity, there has been no scientific need for diabetic animal models. A greater challenge is the mitogenicity already inherent with regular insulin. Thus, the focus for preclinical safety evaluation of analogues is to demonstrate that modifications in regular insulin do not result in enhanced mitogenicity. The approaches used to assess the mitogenic potential of insulin analogues have changed over time driven by scientific progression and changes within the regulatory environment. Therefore, in vitro and in vivo evaluation of cell proliferation has become common practice, and to date there has been no evidence that the mitogenic potential of insulin analogues may be increased compared to regular insulin.