Fig 1.
Insulin administration increases AKT thr308 phosphorylation in BV2 cells.
Insulin (0.36uM) was administered 1 hour after LPS to BV2 cells and AKT phosphorylation at thr308 or ser473 was assessed 2 hours later. A) Insulin alone significantly increased AKT thr308 phosphorylation. LPS also led to an increase in phosphorylation, but this was not significantly different than the control group. Insulin in addition to LPS led to no significant change in AKT phosphorylation from control or the LPS alone group. *p<0.05, two-way ANOVA. B) Western blot for all samples. C) Phosphorylation of Akt at ser473 was markedly different after insulin and LPS treatment, wherein insulin resulted in a slight but not statistically significant elevation, but LPS significantly elevated phosphorylation. ***p<0.001, two-way ANOVA. N = 3/group. Bars represent mean +/- SEM. D) Representative western blot for 2 of 3 samples for ser473.
Fig 2.
Insulin treatment significantly reduces NO and ROS production.
BV2 microglia incubated with LPS showed a significant increase in NO production (A). Insulin treatment after LPS was able to significantly reduce NO production in BV2 microglia. Insulin alone did not significantly alter NO release compared to control cells (0μM insulin) at any concentration. B) BV2 microglia incubated with LPS showed a significant increase in ROS production. Insulin treatment after LPS was able to significantly reduce ROS production in BV2 microglia. Insulin alone did not significantly alter ROS release compared to control cells (0μM insulin) at any concentration. All experimental conditions were repeated in triplicate. *p < 0.05; **p<0.01; ***p<0.001; ****p<0.0001. Bars are mean +/- SEM.
Fig 3.
Insulin treatment reduces the expression of iNOS, but not other pro-inflammatory protein markers.
Western blot quantitation of CD86 showed no significant difference with LPS or insulin administration (A; n = 3/group). LPS exposure resulted in a non-significant increase in CD86 expression. B) Representative blot of CD86 and GAPDH. Western blot quantitation of iNOS showed a significant elevation in iNOS with LPS administration, and this was significantly blunted by insulin (C, n = 3/group). Blots of the quantified bands are shown in D, with ladder for appropriate band sizing shown at each edge. Quantitation of iNOS immunocytochemistry normalized to DAPI expression showed a significant increase with LPS (E), but, while the image shows a slight reduction iNOS intensity (F), this was not reflected in pixel density quantitation (n = 5-6/group). Western blot quantitation of NOX2 showed a significant elevation with LPS administration, this was not significantly blunted by insulin (G, n = 3/group). Blots of the quantified bands are shown in H. Bars are mean +/- SEM. *p<0.05; **p<0.01; ****p<0.0001. Size bar = 50μm.
Fig 4.
Insulin treatment does not alter expression of anti-inflammatory markers YM1 and CD206.
Quantitation of western blots for YM1 showed that LPS exposure significantly decreased expression, but insulin had no effect (n = 3/group, A). Representative bands of YM1 and GAPDH are displayed (B). Quantitation of CD206 immunocytochemistry and normalization to DAPI expression demonstrated no significant effect by any treatment (C). Representative images are shown in D. Bars are mean +/- SEM. *p<0.05. Size bar = 50μm.
Fig 5.
Insulin treatment reduces TNFα.
Analysis of chemoattractant and cytokines known to be affected by LPS and insulin showed that, while LPS significantly reduced IL8 production, insulin did not significantly alter this expression. However, insulin did significantly reduce TNFα expression in comparison to the LPS-treated cells. *p<0.05, ****p<0.0001. n = 3/group. Bars represent mean +/- SEM.
Fig 6.
Insulin treatment alters phagocytic activity.
A) Latex beads conjugated with FITC-IgG (green) were incubated for 4 hours with BV2 cells (DAPI nuclear stain–blue) exposed to control conditions, insulin, LPS, or LPS + insulin (representative images). B) Quantitation of phagocytosed beads demonstrated that insulin alone led to a significant reduction in phagocytosis activity at 28 hours (24 hour incubation with stimulation, 4 hour incubation with beads), which was further reduced by LPS stimulation. Addition of insulin to the LPS treated group partially alleviated this suppression, significantly increasing phagocytosis in comparison to the LPS group. n = 5/group. C) Vybrant phagocytosis assay performed 3 hours after stimulation (2 hours with phagocytic material), showed that acute insulin administration significantly increased phagocytosis. Similar to the longer condition, LPS reduced phagocytosis, which was reversed by insulin administration. TNFa, on the other hand, significantly increased phagocytosis, which was reversed by insulin. N = 3/group. Bars are mean +/- SEM. *p<0.05, **p<0.01, ****p<0.0001. Size bar = 50μm.