Acute high glucose enhanced AKT phosphorylation and attenuated eNOS phosphorylation in HUVECs
PI3K/AKT/eNOS is one of the two parallel pathways activated buy insulin after binding to its receptor [5]. Particularly, AKT is an important signaling molecule that is involved in different endothelial functions, for example the regulation of angiogenesis, proliferation, vascular permeability, survival and cellular transformation as well [17]. Also AKT may phosphorylate eNOS to promote vasodilation with increased NO production in endothelial cells [5].
It has been shown that impaired PI3K/AKT signaling due to hyperglycemia may promote endothelial dysfunction in diabetes [18]. To this aim, we firstly investigated the effects of acute HG in AKT phosphorylation and in its effector eNOS in HUVECs. In high glucose, phosphorylation levels of Ser473AKT increased, while phosphorylation levels of Ser1177eNOS reduced versus the control. The total amount of AKT and eNOS protein levels remained unchanged (Fig. 1). These results suggest that acute HG impairs AKT/eNOS signal transduction.
Time course of insulin-mediated eNOS phosphorylation at Ser1177 in HUVECs
In order to establish the optimal amount of time for insulin incubation, confluent HUVECs were treated with 100 pmol/L insulin at three different time points: 5, 10 and 30 min. The insulin concentration used was 100 pmol/L insulin because it corresponds to physiological plasma insulin levels in humans, so this amount may be considered a good in vitro representation of in vivo insulin activity [16]. After insulin treatment, cells were collected and eNOS phosphorylation in Ser1177 levels were analyzed by Western Blot. We observed gradually increasing levels of Ser1177eNOS between 5 and 30 min, reaching statistical significance at 30 min (Fig. 2).
Physiological insulin administration enhanced AKT and eNOS phosphorylation in NG in a concentration-dependent manner, but had no effects in HG in HUVECs
To investigate the alterations on the PI3K/AKT/eNOS-dependent insulin signaling pathway under HG conditions, activation of AKT and eNOS was determined by immunoblotting. Insulin administered at different physiological concentrations under NG conditions caused a concentration-dependent increase in phospho-Ser473AKT, which was statistically significant at the concentration of 10−9 and 10−8 M (Fig. 3). Contrary to what happened under NG, insulin physiological treatment under HG conditions did not cause the same effect.
To determine whether such a difference in endothelial AKT activity in response to insulin concentrations under the two conditions of NG and HG also had downstream consequences, we next evaluated the phosphorylation of eNOS. The different physiological insulin concentrations added at NG induced an increase in phospho-Ser1177eNOS that nicely correlated to a dose between 10−10 and 10−9 M, but was attenuated at 10−8 M. This could indicate a desensitization of the metabolic insulin signaling pathway. However, as the same insulin concentration continued to increase phosphorylated AKT levels, this indicated that insulin preserved some ability to signal to the metabolic arm.
Interestingly, physiological insulin added to HUVECs acutely treated under high glucose conditions did not follow the same eNOS phosphorylation profile that we observed in NG, indicating that insulin could not prevent high glucose-dependent inhibition of eNOS activation.
Acute high glucose had no effects on ERK1/2, JNK and p38 phosphorylation levels in HUVECs, and physiological insulin treatment increased them under NG and HG conditions
To investigate the alterations in the Ras/Raf/MAPK-dependent insulin signaling pathway in HG, activation of ERK1/2, p38 and JNK was determined. In NG, HUVECs responded to insulin stimulation with an increase in ERK1/2 phosphorylation in a concentration-dependent manner, being statistically significant for insulin doses 10−9 and 10−8 M. We obtained the same results for phospho-p38, which was statistically significant at insulin doses of 10−8 M, and phospho-JNK, with a significant increase at the insulin concentrations of 10−9 and 10−8 M (p < 0.05). Conversely to AKT and eNOS phosphorylation, 24 h of HG treatment did not affect the phosphorylated forms of ERK1/2, p38 or JNK. Intriguingly, insulin physiological treatment in HG maintained an increase of these forms in a concentration-dependent manner, and this increase was significant (1) at all insulin doses tested for phospho-ERK1/2, (2) at the concentration of 10−8 M for phospho-p38 and (3) at the concentrations of 10−9 and 10−8 M for phospho-JNK (Fig. 4).
Acute high glucose attenuated IRβ expression in HUVECs. Insulin administration had no effects under NG and HG in terms of total amount but increased its phosphorylated form
We hypothesized that exposure of HUVECs to HG could affect the early steps of insulin signaling. To test this, IRβ protein expression levels were examined. Acute exposure of HUVECs to HG induced a significant decrease in IRβ tyrosine phosphorylation accompanied by a reduction in IRβ total protein versus the control (Fig. 5).
Insulin treatment under NG conditions enhanced in a dose-dependent manner IRβ tyrosine phosphorylation at all tested physiological insulin concentrations, but it had no effects under HG in HUVECs, although a statistically insignificant increase was observed. Insulin stimulation in NG had no effects on IRβ total expression compared with the untreated control. The decrease in IRβ total expression observed under HG conditions was not counteracted by any tested dose of insulin (Fig. 5).