Fig 1.
Effects of chronic HG on the insulin signaling pathway of HGEC in the presence and absence of insulin pulse (15 min treatment).
HG results in downregulation of the expression (A; Western blot and densitometric analysis) and activation levels of IR (B; Western blot and densitometric analyses) and the expression levels of IRS-1 (C; Western blot and densitometric analysis). HG results in downregulation of tyrosine residue phosphorylation and upregulation of serine residue phosphorylation in IRS-1 (D; Western blot and densitometric analysis). Data represent mean±SEM, n = 3–5, *P<0.05, **P<0.01 vs. corresponding control (5 mmol/L glucose) and •P<0.05 vs. 25 mmol/L glucose control.
Fig 2.
Effects of chronic HG on the downstream insulin signaling pathway of HGEC in the presence and absence of insulin pulse (15 min treatment).
HG results in downregulation of the phosphorylation levels of Akt and Fox01,03a (A; Western blot and densitometric analysis). Insulin action on p-Akt and p-Fox01,03a is PI3K-dependent, as pretreatment of HGEC for 24 h with the PI3K inhibitor wortmannin, abolished the stimulatory effects of insulin (15 min treatment) on p-Akt (B; Western blot and densitometric analysis). Data represent mean±SEM, n = 3, **P<0.01 vs. control (5 mmol/L glucose) and ••P<0.01 vs. control (25 mmol/L glucose).
Fig 3.
Effects of glucose concentration alterations and prolonged insulin treatment on HGEC.
Transition from high to normal glucose for 30 d partially restores p-Akt levels and its response to insulin pulse (A; Western blot and densitometric analysis), while 48 h are enough to partially restore p-IR levels in the presence of insulin (15 min treatment) (B; Western blot and densitometric analysis). Transition from normal to high glucose for 48 h further potentiates IR phosphorylation in response to insulin (B). Prolonged insulin treatment (300 nmol/L Levemir for 24 h) resulted in downregulation of IR levels without affecting its phorsphorylation capacity or p-Akt levels (C; Western blot and densitometric analysis). Densitometric analyses for p-Akt and p-IR are not shown. Data represent mean±SEM, n = 3, *P<0.05, **P<0.01 vs. control (5 mmol/L glucose) and ••P<0.01 vs. control (25 mmol/L glucose). SG; starting glucose, GR; glucose replacement.
Fig 4.
HG predisposes HGEC to apoptosis.
Treatment of HGEC with DTT for 24 h results in increased PARP and Caspase-3 cleavage, a response that occurs in a greater extent in adherent 25 mmol/L glucose-culture cells than in control (5 mmol/L glucose-cultured cells) (A; Western blot image and densitometric analysis; FL-floating cells, AT-attached cells). Increased apoptosis is further confirmed by DNA fragmentation analysis of the floating cells under these treatments (B). TUNEL assay shows apoptosis in HG-treated cells, in the absence of DTT, where DAPI (blue) and TUNEL (red) stain nuclei of viable and apoptotic cells, respectively (C). Data represent mean±SEM, n = 3–5, **P<0.01 vs. control (5 mmol/L glucose); •P<0.05, ••P<0.01 vs. control (25 mmol/L glucose) and #P<0.05 vs. 5mmol/L-DTT treated cells.
Fig 5.
Effects of HG (4d treatment) on the insulin signaling pathway and the survival of isolated glomeruli.
Treatment of glomeruli with 25 mmol/L glucose for 96 h resulted in downregulation of the phosphorylated levels of Akt and IR, without affecting total IR levels (A; Western blot image and densitometric analysis), which coincided with enhanced apoptosis as evident by increased PARP and Casp3 cleavage (B; Western blot image and densitometric analysis). Data represent mean±SEM, n = 3–5, *P<0.05, **P<0.01, ***P<0.001 vs. control (5 mmol/L glucose) and •••P<0.001 vs. 25 mmol/L glucose-treated glomeruli.
Fig 6.
Impaired insulin signaling in the diabetic podocyte results in apoptotic death.
HG causes downregulation of IR, IRS-1 levels, increased phosphorylation of IRS-1 at Ser636 and decreased phosphorylation of Akt and Fox01,03a. All these effects synergize to yield an apoptotic outcome. Abbreviations: IR: Insulin Receptor; IRS: Insulin receptor substrate; PI3K: phosphoinositide 3-kinase; Akt: (protein kinase B); PARP: poly (ADP-ribose) polymerase; Fox01,03: forkhead box protein 01, 03.