Functional coupling of angiotensin II type 1 receptor with insulin resistance of energy substrate uptakes in immortalized cardiomyocytes (HL-1 cells)

Abstract

Background and purpose: Increased angiotensin II levels and insulin resistance coexist at the early stages of cardiomyopathies. To determine whether angiotensin II increases insulin resistance in cardiomyocytes, we studied the effect of angiotensin II on basal and insulin-stimulated transport rate of energy substrates in immortalized cardiomyocytes (HL-1 cells).

Experimental approach: Glucose and palmitic acid uptakes were measured using [(3)H]2-deoxy-D-glucose and [(14)C]palmitic acid, respectively, in cells exposed or not exposed to angiotensin II (100 nM), angiotensin II plus irbesartan or PD123319, type 1 and 2 receptor antagonists, or PD98059, an inhibitor of ERK1/2 activation. Cell viability, DNA, protein synthesis and surface area were evaluated by the MTT test, [(3)H]thymydine, [(3)H]leucine and morphometric analysis, respectively. Type 1 receptor levels were measured by western blot analysis.

Key results: Basal uptakes of glucose and palmitic acid by HL-1 cells (0.37+/-0.07 and 7.31+/-0.22 pmol per 10(4)cells per min, respectively) were both stimulated by 100 nM insulin (+91 and +64%, respectively). Cells exposed to angiotensin II remained viable and did not show signs of hypertrophy. In these conditions, the basal palmitic acid uptake of the cells increased (11.41+/-0.46 pmol per 10(4) cells per min) and insulin failed to stimulate the uptake of glucose and fatty acids. Changes in the rate of uptake of energy substrates were prevented or significantly reduced by irbesartan or PD98059.

Conclusions and implications: Angiotensin II is a candidate for increasing insulin resistance in cardiomyocytes. Our results suggest a further mechanism for the cardiovascular protection offered by the angiotensin II type 1 receptor blockers.

Figure 1

The insulin-stimulated glucose and palmitate transport in (immortalized cardiomyocytes) HL-1 cells. (a) Glucose and palmitate uptakes were measured radiochemically as described in Methods. Insulin stimulates in a concentration-dependent manner the rate of both transports. Insulin potency (EC₅₀) was calculated by the fitting of experimental points. (b) Pre-incubation of cells with wortmannin reduced the insulin stimulation of glucose and palmitic acid uptake. Results are presented as stimulated rate compared to the basal transport rate, taken as 100, and they represent the mean±s.e.mean of at least eight (a) or six (b) experiments run in triplicate. For the basal uptake values see Table 1. ^*P<0.05 vs insulin effect in the absence of wortmannin.

Figure 2

Pharmacological modulation of angiotensin II-induced insulin resistance of glucose uptake. (a) Concentration-dependent effect of insulin (from 5 to 300 nM) on glucose transport in cells exposed to angiotensin II, angiotensin II plus irbesartan (IRB, 1 μM) or PD98059 (10 μM) or angiotensin II plus PD123319 (1 μM). Results are the mean±s.e.mean of 8–18 experiments run in triplicate and are presented as stimulation of their basal uptake rate (taken as 100). Lines represent fitting of data points. (b) The maximum value of insulin (100 nM)-stimulated uptake (as calculated by fitting curves from (a) and from Figure 1) in HL-1 cells challenged with different compounds. Each column is the mean±s.e.mean of 5–18 experiments run in triplicate. ^**P<0.01, compared to insulin maximum effect in control cells.

Figure 3

Pharmacological modulation of angiotensin II-induced insulin resistance of palmitate uptake. (a) The maximum absolute value of insulin (100 nM)-stimulated uptake (pmol per 10⁴ cells per min) in HL-1 cells challenged with different compounds is presented. Each column is the mean±s.e.mean of 5–18 experiments run in triplicate. ^**P<0.01 and ^*P<0.05 compared to uptake in control cells, and ^§P<0.05 compared to insulin effect in control cells. (b) Concentration-dependent stimulation of palmitic acid transport induced by insulin (from 5 to 300 nM) was evaluated radiochemically, as described in Methods, in cells exposed to angiotensin II, angiotensin II plus irbesartan (IRB, 1 μM) or PD98059 (10 μM) or angiotensin II plus PD123319 (1 μM). Results are the mean±s.e.mean of 5–18 experiments run in triplicate and are presented as arbitrary units (see text for details). Lines represent fitting of data points.

Figure 4

Angiotensin II type 1 receptor (AT₁ receptor) levels and evaluation of hypertrophy in HL-1 cells exposed to angiotensin II. (a) Western blot analysis of AT₁ receptors in lysates (30 μg of cell proteins) from control and angiotensin II-treated HL-1 cells. A representative experiment is shown, repeated twice with similar results. (b) DNA and protein labelled with [³H]thymidine and [³H]leucine were evaluated in cells not exposed (control) and exposed to angiotensin II (100 nM) as described in Methods. Results are expressed as counts per minute (CPM) per well and represent the mean±s.e.mean of three experiments run in triplicate. (c) Cell surface area was measured as described in Methods. The means±s.e.mean of the area of 80–120 cells per field analysed are presented.