Protein kinase C-β contributes to impaired endothelial insulin signaling in humans with diabetes mellitus

Abstract

Background: Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling, through the activity of protein kinase C-β (PKCβ) and nuclear factor κB, reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus.

Methods and results: We measured protein expression and insulin response in freshly isolated endothelial cells from patients with type 2 diabetes mellitus (n=40) and nondiabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes mellitus (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in nondiabetic subjects but not in diabetic patients (P=0.003), consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients, indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=-0.541, P=0.02). Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes mellitus. Endothelial nuclear factor κB activation was higher in diabetes mellitus and was reduced with PKCβ inhibition.

Conclusions: We provide evidence for the presence of altered eNOS activation, reduced insulin action, and inflammatory activation in the endothelium of patients with diabetes mellitus. Our findings implicate PKCβ activity in endothelial insulin resistance.

Figure 1

Diabetes mellitus is associated with altered eNOS activation and insulin resistance in endothelial cells. As described in Methods, freshly isolated endothelial cells identified by vWF staining (green) and nuclear morphology (DAPI in blue) were obtained from patients with diabetes (n=12) and non-diabetic controls (n=9). Cells were incubated with 0nM or 10nM insulin for 30 minutes and then fixed. eNOS activation was evaluated as eNOS phosphorylation at serine 1177 (red). Protein levels were quantified by evaluating 20 cells for each patient under each condition. A. Representative cells from a patient with diabetes (right) show higher basal eNOS phosphorylation at serine 1177 (top, red) compared to the non-diabetic control (left top, red). Insulin increased eNOS phosphorylation at serine 1177 in the non-diabetic control (left top, red vs. left bottom, red) but not in the diabetic patient (right top, red vs. right bottom, red). B. Pooled data show that basal eNOS phosphorylation at serine 1177 was 67% higher in endothelial cells from patients with diabetes compared to non-diabetic controls (*P<0.01). C. Pooled data demonstrate that insulin increased eNOS phosphorylation at serine 1177 in endothelial cells from non-diabetic controls but not in endothelial cells from patients with diabetes (*P=0.003).

Figure 2

Diabetes mellitus and inhibitory eNOS phosphorylation. As described in Methods, freshly isolated endothelial cells identified by vWF staining (green) and nuclear morphology (DAPI in blue) were obtained from patients with diabetes (n=9) and non-diabetic controls (n=6). Cells were incubated with 0nM or 10nM insulin for 30 minutes and then fixed. eNOS phosphorylation at threonine 495 (red) was quantified by evaluating 20 cells for each patient under each condition. A. Representative cells from a patient with diabetes (right) show similar basal eNOS phosphorylation at threonine 495 (top, red) compared to the non-diabetic control (left top, red). Insulin decreased eNOS phosphorylation at threonine 495 in the non-diabetic control (left top, red vs. left bottom, red) and increased phosphorylation at this site in the diabetic patient (right top, red vs. right bottom, red). B. Pooled data show that basal eNOS phosphorylation at threonine 495 was similar in endothelial cells from patients with diabetes compared to non-diabetic controls (P=0.95). C. Pooled data demonstrate that insulin decreased eNOS phosphorylation at threonine 495 in endothelial cells from non-diabetic controls but increased in endothelial cells from patients with diabetes (*P=0.009).

Figure 3

Abnormal eNOS activation is not attributable to differential eNOS, Akt or PTEN expression in diabetes. Diabetes is associated with oxidative stress in endothelial cells. Freshly isolated endothelial cells collected from patients with diabetes and non-diabetic controls were fixed and stained (20 cells per patient for each protein). A. Representative cell from a diabetic patient (right) shows similar eNOS expression (red) compared with a cell from a non-diabetic control (left, red). Pooled data show that eNOS levels were comparable in the non-diabetic controls (n=8) and diabetics (n=10, P=0.46). B. Representative cell from a diabetic patient (right) shows similar Akt expression (red) compared with a cell from a non-diabetic control (left, red). Pooled data show that Akt levels were comparable in the non-diabetic controls (n=6) and diabetics (n=10, P=0.79). C. Representative cell from a diabetic patient (right) shows similar PTEN expression (red) compared with a cell from a non-diabetic control (left, red). Pooled data show that PTEN levels were comparable in the non-diabetic controls (n=8) and diabetics (n=10, P=1.0). D. Representative cell from a diabetic patient (right) shows higher nitrotyrosine expression (red) compared with a cell from a non-diabetic control (left, red). Pooled data show that nitrotyrosine levels were higher diabetic patients (n=5) compared to the non-diabetic controls (n=5, *P=0.03).

Figure 4

PKCβ and Endothelial Function. A. Representative cell from a diabetic patient (right) shows higher PKCβ expression (red) compared with a cell from a non-diabetic control (left, red). Pooled data (20 cells analyzed per patient) show that PKCβ levels were higher in diabetic patients (n=11) compared to the non-diabetic controls (n=7, *P=0.03). B. Higher endothelial PKCβ expression levels were associated with lower flow-mediated dilation.

Figure 5

Inhibition of PKCβ alters eNOS phosphorylation and insulin response in endothelial cells from diabetic patients. As described in Methods, freshly isolated endothelial cells from patients with diabetes (n=7) were treated with 0 or 30nM LY379196, a pharmacologic inhibitor of PKCβ, along with 0 or 10nM insulin for 30 minutes and eNOS phosphorylation at serine 1177 was measured (20 cells per patient in each condition). A. Representative cells from a patient with diabetes treated with LY379196 (right) shows lower basal eNOS phosphorylation at serine 1177 (top, red) compared to control condition (left top, red). Treatment with LY379196 improved the insulin-mediated change in eNOS phosphorylation at serine 1177 (right top, red vs. right bottom, red) compared to control condition (left top, red vs. left bottom, red). B. Basal eNOS phosphorylation at serine 1177 was reduced in endothelial cells from patients with diabetes treated with LY379196 (*P=0.02). C. Treatment with LY379196 improved insulin-mediated eNOS phosphorylation at serine 1177 in endothelial cells from patients with diabetes (*P=0.04).

Figure 6

PKCβ and endothelial inflammatory activation in diabetes. A. There was a trend for higher expression of NFκB subunit p65 in endothelial cells from patients with diabetes (n=4 diabetic and 5 non-diabetic, *P=0.06). B. There was a trend for higher expression of ICAM-1, a cellular adhesion molecule regulated by NFκB, in endothelial cells from patients with diabetes (n=4 diabetic and 5 non-diabetic, *P=0.06). C. Expression of IκBα, an inhibitor of NFκB, was lower in endothelial cells from patients with diabetes (n=6 diabetic and 5 non-diabetic, *P=0.004). Treatment with LY379196 for 30 minutes increased IκBα expression in patients with diabetes (**P=0.03).