Insulin signaling coordinately regulates cardiac size, metabolism, and contractile protein isoform expression

Abstract

To investigate the role of insulin signaling on postnatal cardiac development, physiology, and cardiac metabolism, we generated mice with a cardiomyocyte-selective insulin receptor knockout (CIRKO) using cre/loxP recombination. Hearts of CIRKO mice were reduced in size by 20-30% due to reduced cardiomyocyte size and had persistent expression of the fetal beta-myosin heavy chain isoform. In CIRKO hearts, glucose transporter 1 (GLUT1) expression was reduced by about 50%, but there was a twofold increase in GLUT4 expression as well as increased rates of cardiac glucose uptake in vivo and increased glycolysis in isolated working hearts. Fatty acid oxidation rates were diminished as a result of reduced expression of enzymes that catalyze mitochondrial beta-oxidation. Although basal rates of glucose oxidation were reduced, insulin unexpectedly stimulated glucose oxidation and glycogenolysis in CIRKO hearts. Cardiac performance in vivo and in isolated hearts was mildly impaired. Thus, insulin signaling plays an important developmental role in regulating postnatal cardiac size, myosin isoform expression, and the switching of cardiac substrate utilization from glucose to fatty acids. Insulin may also modulate cardiac myocyte metabolism through paracrine mechanisms by activating insulin receptors in other cell types within the heart.

Figure 1

Insulin receptor (IR) levels and IR autophosphorylation in CIRKO mice. Upper panels show representative immunoblots blotted for the IR in various tissues of CIRKO mice (right), and homogenates of cardiac ventricles from CIRKO and wild-type (WT) mice (left). Lower panels show a representative immunoblot for the IR from isolated cardiomyocytes obtained from CIRKO (KO) and WT mice (left), and a phosphotyrosine (P-Tyr) immunoprecipitate showing IR phosphorylation in cardiomyocytes from WT and KO mice following insulin stimulation (right). Data are from 8- to 12-week-old male mice and are representative of three to four experiments on separate animals. BAT, brown adipose tissue.

Figure 2

Heart and myocyte size in CIRKO and WT mice. (a) Examples of hearts from 5-week-old male and female CIRKO mice and littermate controls. Ventricular wall thicknesses of the hearts shown are: male WT, 2000 μm; male CIRKO, 800 μm; female WT, 1300 μm; and female CIRKO, 800 μm. (b) Representative photomicrographs of isolated cardiomyocytes obtained from 12-week-old male CIRKO mice. Myocyte dimensions (n = 100 myocytes/mouse ×3 mice) are shown in the table below. Data are means ± SE. *P < 0.0001 vs. CIRKO.

Figure 3

Cardiac histology. Representative transverse sections of left ventricle stained with H&E (×20, upper panels) and Trichrome (×10, lower panels) obtained from CIRKO and WT mice. Upper panels are from 5-week-old males, and lower panels are from 5-week-old females. Both H&E and Trichrome data exist for each sex.

Figure 4

Glucose transporter expression. Upper panels show representative GLUT1 and GLUT4 immunoblots obtained from the hearts of 12-week-old CIRKO and littermate control mice (WT). Lower panels show densitometric analyses of six to nine independent blots. *P < 0.0001, ^†P < 0.04, ^#P < 0.05, and ^§P < 0.01 vs. WT of the same sex. Data are means ± SE.

Figure 5

Glucose transport in isolated cardiomyocytes. Left panel shows 2-deoxyglucose uptake in response to insulin stimulation at the concentrations shown. Right panel shows 2-deoxyglucose uptake in response to IGF-1 stimulation at the concentrations shown. Data are obtained from cardiomyocytes obtained from 12-week-old male CIRKO and age-matched controls and represent three independent experiments performed in triplicate. Data are means ± SE. *P < 0.0001 vs. WT treated with equivalent dose of insulin or IGF-1, **P < 0.004 vs. 1 nM IGF-1, ***P < 0.0005 vs. 10 nM IGF-1, ^†P < 0.0001 vs. basal of same genotype, ^‡P < 0.001 vs. 1 nM IGF-1, ^#P < 0.002 vs. 0.1 nM insulin, ^##P < 0.01 vs. basal of same genotype.

Figure 6

Cardiac metabolism in isolated working hearts, and in vivo glucose uptake in CIRKO and control mice. Average rates of glycolysis, glucose oxidation, fatty acid oxidation, and glycogen content after 60 minutes of perfusion and in vivo cardiac 2-deoxyglucose uptake are shown in the panels as labeled. All studies were performed in 16- to 20-week-old male CIRKO mice and littermate controls. In the isolated heart studies, equal numbers of CIRKO and control mice were studied in each experiment. Numbers of animals studied are as follows. Without insulin: glucose oxidation and glycolysis, n = 5; fatty acid oxidation, n = 4; glycogen, n = 9. With insulin: glucose oxidation and glycolysis, n = 7; fatty acid oxidation, n = 6; glycogen, n = 13. For the in vivo 2-deoxyglucose uptake studies, numbers of mice are n = 4 and 5 (WT, basal and insulin, respectively) and n = 4 and 3 (CIRKO, basal and insulin, respectively). Data are means ± SE. *P < 0.05 vs. basal, ^†P < 0.05 vs. WT.

Figure 7

Gene expression analysis in CIRKO hearts. mRNA levels of MCAD, LCAD, VLCAD, PPARα, PDK4, and β-MHC in hearts of WT and CIRKO mice. Real-time PCR data were obtained from four CIRKO and four WT littermate controls, and for Northern blot analysis from three CIRKO and three WT. Each transcript was analyzed in duplicate. Data are means ± SE. *P < 0.05, **P < 0.0007 vs. WT.