Angiotensin II shifts insulin signaling into vascular remodeling from glucose metabolism in vascular smooth muscle cells

Abstract

Background: To clarify the role of angiotensin II (Ang II) in insulin-induced arteriosclerosis, we examined the effects of Ang II on insulin-induced mitogen-activated protein (MAP) kinase activation and cellular hypertrophy in rat vascular smooth muscle cells (VSMCs).

Methods: Phosphorylated MAP kinases were detected with western blot analysis. Cellular hypertrophy and glucose uptake were evaluated from incorporation of [(3)H]-labeled-leucine and -deoxy-D-glucose, respectively. Cell sizes were measured by Coulter counter.

Results: While Ang II (100 nmol/l, 18 h) augmented cellular hypertrophy by insulin (10 nmol/l, 24 h), insulin alone did not affect hypertrophy without Ang II pretreatment. Insulin increased p38MAP kinase and c-Jun N-terminal kinase (JNK) phosphorylation; in the presence of Ang II, p38MAP kinase, and JNK were further activated by insulin. Treatment of a p38MAP kinase inhibitor, SB203580 (10 µmol/l), and a JNK inhibitor, SP600125 (20 µmol/l), abrogated the [(3)H]-leucine incorporation by insulin in the presence of Ang II. Both the Ang II receptor blocker, RNH-6270 (100 nmol/l), and an antioxidant, ebselen (40 µmol/l), inhibited vascular cell hypertrophy. Specific depletion of insulin receptor substrate-1 with small interfering RNA increased [(3)H]-leucine incorporation by insulin (10 nmol/l, 24 h); pretreatment with Ang II attenuated insulin (10 nmol/l, 30 min)-induced glucose uptake.

Conclusions: Ang II attenuates insulin-stimulated glucose uptake and enhances vascular cell hypertrophy via oxidative stress- and MAP kinase-mediated pathways in VSMCs. Ang II may also cause insulin signaling to diverge from glucose metabolism into vascular remodeling, affecting insulin-induced arteriosclerosis in hypertension.

Figure 1

Effect of IRS-1 siRNA on Akt phosphorylation and protein synthesis. (a) VSMCs were transfected with IRS-1 siRNA or scrambled siRNA for 48 h and then exposed to vehicle or 10 nmol/l insulin for 5 min. Western blotting with anti-IRS-1, phospho-Akt or Akt antibody was performed. (b) VSMCs transfected with IRS-1 siRNA or scrambled siRNA were exposed to vehicle or 10 nmol/l insulin for 24 h. Protein synthesis was measured by [3H]-leucine incorporation. Data represent mean ± s.e. (n = 6), expressed as fold change compared with unstimulated cells. *P < 0.05 vs. control VSMCs. IRS-1, insulin receptor substrate-1; siRNA, small interfering RNA; VSMCs, vascular smooth muscle cells.

Figure 2

Effect of Ang II on insulin-induced MAP kinase phosphorylation. (a,d,f) VSMCs were pretreated with Ang II (100 nmol/l) for 18 h and then exposed to 10 nmol/l insulin for the indicated times. (b,c,e,g) VSMCs were pretreated with vehicle or Ang II (100 nmol/l, 18 h) and then exposed to insulin (10 nmol/l, 5 min). Western blotting was performed with anti-IRS-1, phospho-p38MAP kinase, p38MAP kinase (a,b,c), phospho-JNK, JNK (d,e), and phospho-ERK 1/2, ERK 1/2 (f,g) antibodies. Data represent mean ± s.e. (n = 5), expressed as fold change compared with unstimulated cells. *P < 0.05 vs. control. Ang II, angiotensin II; ERK, extracellular regulated kinase; IRS-1, insulin receptor substrate-1; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; VSMCs, vascular smooth muscle cells.

Figure 3

Effect of Ang II on insulin-induced protein synthesis and cell hypertrophy. (a) VSMCs were pretreated with Ang II (100 nmol/l) for 18 h and then exposed to with the indicated concentrations of insulin for 24 h. (b) Cells were pretreated with MAP kinase inhibitors (SB203580; 10 μmol/l, SP600125; 20 μmol/l, PD98059; 10 μmol/l), wortmannin (100 nmol/l) or an antioxidant (ebselen; 40 μmol/l) before insulin (10 nmol/l) incubation (30 min). The ARB (RNH-6270; 100 nmol/l) pretreated before Ang II incubation (30 min). Protein synthesis was measured by [³H]-leucine incorporation. (c) Cell volume was measured by a Coulter Cell Multisizer. Data represent mean ± s.e. (n = 6), expressed as fold change compared with unstimulated cells. Two-way ANOVA showed that the dose-dependent increase of [³H]-leucine incorporation was significantly higher in Ang II-pretreated VSMCs than in control VSMCs. *P < 0.05 vs. control VSMCs. **P < 0.05 vs. Ang II-pretreated VSMCs without insulin. ^#P < 0.05 vs. insulin treatment in Ang II-pretreated VSMCs. Ang II, angiotensin II; ARB, Ang II type 1 receptor blockers; MAP, mitogen-activated protein; VSMCs, vascular smooth muscle cells.

Figure 4

Effect of Ang II on insulin-induced glucose uptake. VSMCs were treated with 100 nmol/l Ang II or vehicle for 18 h in the presence or absence of an ARB, exposed to 10 nmol/l insulin for 30 min, and 2-deoxy-D-[³H]-glucose uptake measured. Data represent mean ± s.e. (n = 10) of percent change in uptake. *P < 0.05 vs. control. Ang II, angiotensin II; ARB, Ang II type 1 receptor blockers; VSMCs, vascular smooth muscle cells.