Acute effects of euglycemic-hyperinsulinemia on myocardial contractility in male mice

Abstract

Type 2 diabetes and obesity are associated with increased risk of cardiovascular disease, including heart failure. A hallmark of these dysmetabolic states is hyperinsulinemia and decreased cardiac reserve. However, the direct effects of hyperinsulinemia on myocardial function are incompletely understood. In this study, using invasive hemodynamics in mice, we studied the effects of short-term euglycemic hyperinsulinemia on basal myocardial function and subsequent responses of the myocardium to β-adrenergic stimulation. We found that cardiac function as measured by left ventricular (LV) invasive hemodynamics is not influenced by acute exposure to hyperinsulinemia, induced by an intravenous insulin injection with concurrent inotropic stimulation induced by β-adrenergic stimulation secondary to isoproterenol administration. When animals were exposed to 120-min of hyperinsulinemia by euglycemic-hyperinsulinemic clamps, there was a significant decrease in LV developed pressure, perhaps secondary to the systemic vasodilatory effects of insulin. Despite the baseline reduction, the contractile response to β-adrenergic stimulation remained intact in animals subject to euglycemic hyperinsulinemic clamps. β-adrenergic activation of phospholamban phosphorylation was not impaired by hyperinsulinemia. These results suggest that short-term hyperinsulinemia does not impair cardiac inotropic response to β-adrenergic stimulation in vivo.

FIGURE 1

(a) Blood glucose in anesthetized mice injected with intravenous (i.v.) bolus of insulin (1 U/kg). (b) Mean arterial pressure measured in the carotid artery of animals injected with i.v. bolus of saline or insulin. (c) Myocardial insulin levels in animals subject to an i.v. bolus of saline or insulin (1 IU/kg). Data are presented as mean ± SEM. In (a) and (b), data were analyzed by repeated measures two‐way ANOVA followed by post‐hoc analysis using Tukey's test (within group comparisons) or Sidak's test (between group comparisons). Statistical significance was set at p < 0.05 (N > 4 animals/group; within group comparisons—**p < 0.01; ^p < 0.05, ^^^^p < 0.01 vs. saline). In (c), data were analyzed by student t‐test (**p < 0.01). ANOVA, analysis of variance; SEM, standard error of the mean.

FIGURE 2

(a) Time dependent measures of cardiac function as determined by LV invasive hemodynamics in animals subject to intravenous (i.v.) bolus of saline or insulin. Data are presented as mean ± SEM and were analyzed by repeated measures two‐way ANOVA to determine the effect of insulin on cardiac function. Statistical significance was set at p < 0.05 (N > 10 animals/group). (b) Measures of cardiac function as determined by LV invasive hemodynamics in animals subject to i.v. bolus of saline or insulin followed by increasing doses of isoproterenol. Data are presented as mean ± SEM and were analyzed by repeated measures two‐way ANOVA to determine difference in inotropic response between groups. Statistical significance was set at p < 0.05 (N > 10 animals/group) and no significant differences in response to isoproterenol were present between the groups by Sidak's post‐hoc test. ANOVA, analysis of variance; LV, left ventricular; SEM, standard error of the mean.

FIGURE 3

Myocardial phosphorylation of (a) AKT, (b) PLN in mice injected with intravenous (i.v.) bolus of saline or insulin (1 U/kg) and increasing doses of isoproterenol, (c) myocardial phosphorylation of PLN in animals injected with increasing doses of isoproterenol after euglycemic hyperinsulinemic clamps. Quantification of data in western blots is presented in graphs below. Data are presented as mean ± SEM. In (a) and (c) data were analyzed by student t‐ test (**p < 0.01, ****p < 0.0001). PLN, phospholamban; SEM, standard error of the mean.

FIGURE 4

(a) Blood glucose levels, (b) glucose infusion rates and (c) serum insulin levels in animals subject to euglycemic‐hyperinsulinemic clamps. Data are presented as mean ± SEM. In (a), data were analyzed by two‐way repeated measures ANOVA followed by Tukey's post‐hoc test. Statistical significance was set at p < 0.05 (N > 4 animals/group; α, β, γ, Δ—saline vs. insulin, *, ^saline vs. insulin + l‐NAME; α, *p < 0.05, β, p < 0.01, γ, ^p < 0.001, Δ, p < 0.0001). In (c), data were analyzed by repeated measures two‐way ANOVA followed by post‐hoc analysis using Tukey's test (within group comparisons) or Sidak's test (between group comparisons). Statistical significance was set at p < 0.05 (N > 4 animals/group; between group comparisons—**p < 0.01, ****p < 0.0001). ANOVA, analysis of variance; l‐NAME, Nω‐nitro‐l‐arginine methyl ester; SEM, standard error of the mean.

FIGURE 5

Measures of cardiac function as determined by LV invasive hemodynamics in animals subject to euglycemic‐hyperinsulinemic clamp followed by increasing doses of isoproterenol. Data are presented as mean ± SEM and were analyzed by repeated measures two‐way ANOVA to determine difference in inotropic response within and between groups. Statistical significance was set at p < 0.05 (N > 10 animals/group) and no significant differences in response to isoproterenol were present between the groups by Sidak's post‐hoc test. (* ^(t) p < 0.05 by student's t‐test). ANOVA, analysis of variance; LV, left ventricular; SEM, standard error of the mean.