Our results suggest that the deletion of AT2 receptor reduced SHP-1 activity and restored VEGF actions, leading to an increased blood flow reperfusion after ischemia in diabetes mellitus.

Decreased collateral vessel formation in diabetic peripheral limbs is characterized by abnormalities of the angiogenic response to ischemia. Hyperglycemia is known to activate protein kinase C (PKC), affecting the expression and activity of growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The current study investigates the role of PKCδ in diabetes-induced poor collateral vessel formation and inhibition of angiogenic factors expression and actions. Ischemic adductor muscles of diabetic Prkcd+/+ mice exhibited reduced blood reperfusion, vascular density, and number of small vessels compared with nondiabetic Prkcd+/+ mice. By contrast, diabetic Prkcd−/− mice showed significant increased blood flow, capillary density, and number of capillaries. Although expression of various PKC isoforms was unchanged, activation of PKCδ was increased in diabetic Prkcd+/+ mice. VEGF and PDGF mRNA and protein expression were decreased in the muscles of diabetic Prkcd+/+ mice and were normalized in diabetic Prkcd−/− mice. Furthermore, phosphorylation of VEGF receptor 2 (VEGFR2) and PDGF receptor-β (PDGFR-β) were blunted in diabetic Prkcd+/+ mice but elevated in diabetic Prkcd−/− mice. The inhibition of VEGFR2 and PDGFR-β activity was associated with increased SHP-1 expression. In conclusion, our data have uncovered the mechanisms by which PKCδ activation induced poor collateral vessel formation, offering potential novel targets to regulate angiogenesis therapeutically in diabetic patients.

Renal podocyte apoptosis is an early event of diabetic nephropathy progression. Insulin action is critical for podocyte survival. Previous studies demonstrated that Src homology-2 domain-containing phosphatase-1 (SHP-1) is elevated in renal cortex of type 1 diabetic mice; we hypothesized that hyperglycemia-induced SHP-1 expression may affect insulin actions in podocytes. Type 1 diabetic Akita mice (Ins2 ϩ/C96Y ) developed elevated foot process effacement and podocyte apoptosis compared with control littermate mice (Ins2 ϩ/ϩ ). In contrast to Ins2 ϩ/ϩ mice, insulin-stimulated protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) phosphorylation were remarkably reduced in renal podocytes of Akita mice. This renal insulin resistance was associated with elevated SHP-1 expression in the glomeruli. Cultured podocytes exposed to high glucose concentration (HG; 25 mM) for 96 h exhibited high levels of apoptotic markers and caspase-3/7 enzymatic activity. HG exposure raised mRNA and protein levels of SHP-1 and reduced the insulin-signaling pathway in podocytes. Overexpression of dominant-negative SHP-1 in podocytes prevented HG effects and restored insulin actions. Elevated SHP-1 expression induced by high glucose levels was directly associated with insulin receptor-␤ in vitro and in vivo to prevent insulin-stimulated Akt and ERK phosphorylation. In conclusion, our results showed that high levels of SHP-1 expression in glomeruli cause insulin resistance and podocyte loss, thereby contributing to diabetic nephropathy. insulin signaling; protein tyrosine phosphatase; insulin receptor-␤; Src homology-2 domain-containing phosphatase-1

Poor glycemic control profoundly affects protein expression and the cell signaling action that contributes to glycemic memory and irreversible progression of diabetic nephropathy (DN). We demonstrate that SHP-1 is elevated in podocytes of diabetic mice, causing insulin unresponsiveness and DN. Thus, sustained SHP-1 expression caused by hyperglycemia despite systemic glucose normalization could contribute to the glycemic memory effect in DN. Microalbuminuria, glomerular filtration rate, mesangial cell expansion, and collagen type IV and transforming growth factor-β expression were significantly increased in diabetic Ins2 mice compared with nondiabetic Ins2 mice and remained elevated despite glucose normalization with insulin implants. A persistent increase of SHP-1 expression in podocytes despite normalization of systemic glucose levels was associated with sustained inhibition of the insulin signaling pathways. In cultured podocytes, high glucose levels increased mRNA, protein expression, and phosphatase activity of SHP-1, which remained elevated despite glucose concentration returning to normal, causing persistent insulin receptor-β inhibition. Histone posttranslational modification analysis showed that the promoter region of SHP-1 was enriched with H3K4me1 and H3K9/14ac in diabetic glomeruli and podocytes, which remained elevated despite glucose level normalization. Hyperglycemia induces SHP-1 promoter epigenetic modifications, causing its persistent expression and activity and leading to insulin resistance, podocyte dysfunction, and DN.