Insulin signaling in the heart is impaired by growth hormone: a direct and early event

Abstract

Growth hormone (GH) exerts major actions in cardiac growth and metabolism. Considering the important role of insulin in the heart and the well-established anti-insulin effects of GH, cardiac insulin resistance may play a role in the cardiopathology observed in acromegalic patients. As conditions of prolonged exposure to GH are associated with a concomitant increase of circulating GH, IGF1 and insulin levels, to dissect the direct effects of GH, in this study, we evaluated the activation of insulin signaling in the heart using four different models: (i) transgenic mice overexpressing GH, with chronically elevated GH, IGF1 and insulin circulating levels; (ii) liver IGF1-deficient mice, with chronically elevated GH and insulin but decreased IGF1 circulating levels; (iii) mice treated with GH for a short period of time; (iv) primary culture of rat cardiomyocytes incubated with GH. Despite the differences in the development of cardiomegaly and in the metabolic alterations among the three experimental mouse models analyzed, exposure to GH was consistently associated with a decreased response to acute insulin stimulation in the heart at the receptor level and through the PI3K/AKT pathway. Moreover, a blunted response to insulin stimulation of this signaling pathway was also observed in cultured cardiomyocytes of neonatal rats incubated with GH. Therefore, the key novel finding of this work is that impairment of insulin signaling in the heart is a direct and early event observed as a consequence of exposure to GH, which may play a major role in the development of cardiac pathology.

Keywords: GH; heart; insulin; signaling.

Figure 1.. Activation of insulin signaling in the heart of GH-Tg mice.

Normal (N) and GH-Tg (T) female mice were injected with saline (−) or insulin (+) and the heart was removed after 5 min. Equal amounts of solubilized heart protein were subjected to immunoblotting using specific antibodies to detect IR phosphorylation at Y⁹⁷² and protein content (A), IRS1/2 phosphorylation at Y⁶¹² and IRS1 protein content (B), Akt phosphorylation at T³⁰⁸ and protein content (C), GSK3β phosphorylation at S⁹ and protein content (D), AS160 phosphorylation at T⁶⁴² and protein content (E) and mTOR phosphorylation at S²⁴⁴⁸ and protein content (F). β-tubulin was used to control protein loading. Data are the mean ± SEM of five to seven different individuals per group. Groups denoted by different letters are significantly different (P<0.05), assessed by two-way ANOVA followed by Bonferroni post-test. NS: not significant. Representative immunoblots are shown.

Figure 2.. Basal phosphorylation of insulin signaling mediators in the heart of GH-Tg mice.

Equal amounts of solubilized heart protein from normal (N) and GH-Tg (T) female mice were subjected to immunoblotting using specific antibodies to detect the phosphorylation of IR at Y⁹⁷² (A), IRS1/2 at Y⁶¹² (B), Akt at T³⁰⁸ (C), GSK3β at S⁹ (D), AS160 at T⁶⁴² (E) and mTOR at S²⁴⁴⁸ (F). β-tubulin was used to control protein loading. Data are the mean ± SEM of six to seven different individuals per group. Groups denoted by different letters are significantly different (P<0.05), assessed by Student’s t test. NS: not significant. Representative immunoblots are shown. Discontinuous lanes in the image indicate blot splicing, performed when the most representative samples of the two experimental groups shown in the graph were not run in contiguous lanes

Figure 3.. Activation of insulin signaling in the heart of LID mice.

Normal (N) and LID (L) female mice were injected with saline (−) or insulin (+) and the heart was removed after 5 min. Equal amounts of solubilized heart protein were subjected to immunoblotting using specific antibodies to detect IR phosphorylation at Y⁹⁷² and Y¹¹⁵⁸/Y¹¹⁶²/Y¹¹⁶³ and protein content (A), IRS1/2 phosphorylation at Y⁶¹² and IRS1 protein content (B), Akt phosphorylation at S⁴⁷³ and T³⁰⁸ and protein content (C), GSK3β phosphorylation at S⁹ and protein content (D), AS160 phosphorylation at T⁶⁴² and protein content (E) and mTOR phosphorylation at S²⁴⁴⁸ and protein content (F). β-tubulin or Coomassie blue staining (CBS) of PVDF membranes were used to control protein loading. Data are the mean ± SEM of seven to eleven different individuals per group. Groups denoted by different letters are significantly different (P<0.05), assessed by two-way ANOVA followed by Bonferroni post-test. NS: not significant. Representative immunoblots are shown.

Figure 4.. Activation of insulin signaling in the heart of GH-treated mice.

Female Swiss-Webster mice were treated with GH (2 mg/kg/day administered in two daily subcutaneous injections) for four days, control animals received saline solution (−). Six h after the last GH administration, mice were injected with saline (−) or insulin (Ins) and the heart was removed after 5 min. Equal amounts of solubilized heart protein were subjected to immunoblotting using specific antibodies to detect IR phosphorylation at Y⁹⁷² and protein content (A), IRS1/2 phosphorylation at Y⁶¹² and IRS1 protein content (B), Akt phosphorylation at T³⁰⁸ and protein content (C), GSK3β phosphorylation at S⁹ and protein content (D), AS160 phosphorylation at T⁶⁴² and protein content (E) and mTOR phosphorylation at S²⁴⁴⁸ and protein content (F). β-tubulin was used to control protein loading. Data are the mean ± SEM of five to eight different individuals per group. Groups denoted by different letters are significantly different (P<0.05), assessed by two-way ANOVA followed by Bonferroni post-test. NS: not significant. Representative immunoblots are shown. −/−: control saline treated mice, acute injection with saline; −/Ins: control saline treated mice, acute injection with insulin; GH/−: GH-treated mice, acute injection with saline; GH/Ins: GH-treated mice, acute injection with insulin.

Figure 5.. Basal phosphorylation of insulin signaling mediators in the heart of LID and GH-treated mice.

Basal phosphorylation of insulin signaling mediators in the heart of normal (N) and LID (L) female mice is depicted in panels A to F. Results for female Swiss-Webster mice treated with GH (2 mg/kg/day in two daily subcutaneous injections) for four days, and the corresponding control animals that received saline (−), are shown in panels G to L. Equal amounts of solubilized heart protein were subjected to immunoblotting using specific antibodies to detect the phosphorylation of IR at Y⁹⁷² (A, G), IRS1/2 at Y⁶¹² (B, H), Akt at S⁴⁷³ (C, I), GSK3β at S⁹ (D, J), AS160 at T⁶⁴² (E, K) and mTOR at S²⁴⁴⁸ (F, L), as well as their protein content. β-tubulin, β-actin or Coomassie blue staining (CBS) of PVDF membranes were used to control protein loading. Data are the mean ± SEM of six to eleven different individuals per group. Groups denoted by different letters are significantly different (P<0.05), assessed by Student’s t test. NS: not significant. Representative immunoblots are shown. Discontinuous lanes in the image indicate blot splicing, performed when the most representative samples of the two experimental groups shown in the graph were not run in contiguous lanes

Figure 6.. Basal phosphorylation and protein content of p38 and Erk1/2 in the heart of GH-Tg, LID and GH-treated mice.

Results corresponding to GH-Tg (T) and their normal controls (N) are shown in panels A and B; those from LID (L) and their normal controls (N) are displayed in panels C and D. Results for female Swiss-Webster mice treated with GH (2 mg/kg/day in two daily subcutaneous injections) for four days, and the corresponding control animals that received saline (−), are shown in panels E and F. Equal amounts of solubilized heart protein from female mice were subjected to immunoblotting using specific antibodies to detect the phosphorylation of Erk1/2 at T²⁰²Y²⁰⁴ and its protein content (A, C, E), and the phosphorylation of p38 at T¹⁸⁰Y¹⁸² (B, D, F). β-tubulin or β-actin were used to control protein loading. Data are the mean ± SEM of five to seven (A-B), ten to eleven (C-D) and nine to ten (E-F) different individuals per group. Groups denoted by different letters are significantly different (P<0.05), assessed by Student’s t test. NS: not significant. Representative immunoblots are shown.

Figure 7.. IRS1 serine phosphorylation and SOCS3 and GLUT4 expression in the heart of GH-Tg, LID and GH-treated mice.

The serine phosphorylation of IRS1 and the mRNA content of SOCS3 and GLUT4 were assessed in female GH-Tg (T), LID (L) mice and their normal controls (N), and in Swiss-Webster mice treated with GH (2 mg/kg/day in two daily subcutaneous injections) for four days, and the corresponding control animals that received saline (−). Equal amounts of solubilized heart protein were subjected to an ELISA assay to determine IRS1 phosphorylation at S³⁰⁷ in GH-Tg (A), LID (B) and GH-treated mice (C). For GH-treated mice, IRS1 phosphorylation at S^636/639 was also determined by immunoblotting, as well as its protein content (D). SOCS3 expression was assessed by RT-qPCR in GH-Tg (E), LID (F) and GH-treated mice (G). GLUT4 expression was evaluated by RT-qPCR in GH-Tg (H), LID (I) and GH-treated mice (J), and by the determination of its protein content by immunblotting in LID (K) and GH-treated mice (L). β-actin or β-tubulin were used to control protein loading, representative immunoblots are shown. The target gene relative expression mRNA levels in RT-qPCR experiments were normalized by the geometric mean of four housekeeping genes: Cyclophilin A, 18 S ribosomal RNA, β-actin, and β-2 microglobulin. Data are the mean ± SEM of different individuals per group (6 for ELISA for GH-Tg mice, 15 to 20 for ELISA for LID and GH-treated mice, 5 to 10 for immunoblotting and 10 for RT-qPCR). Groups denoted by different letters are significantly different (P<0.05), assessed by Student’s t test. NS: not significant.

Figure 8.. GHR, IGF-1R and IGF-1 expression in the heart of GH-Tg, LID and GH-treated mice.

The cardiac expression of GHR, IGF-1R and IGF-1 was assessed by RT-qPCR in female GH-Tg (T), LID (L) mice and their normal controls (N), and in Swiss-Webster mice treated with GH (2 mg/kg/day in two daily subcutaneous injections) for four days, and the corresponding control animals that received saline (−). The target gene relative expression mRNA levels were normalized by the geometric mean of four housekeeping genes: Cyclophilin A, 18 S ribosomal RNA, β-actin and β-2 microglobulin. Data are the mean ± SEM of seven to eleven different individuals per group. Groups denoted by different letters are significantly different (P<0.05), assessed by Student’s t test. NS: not significant.

Figure 9.. Activation of insulin signaling in primary culture of rat cardiomyocytes incubated with GH.

Cardiac myocytes were incubated with GH (1 μg/mL) for 24 h prior to stimulation with insulin (10⁻⁵ M) for 10 min. Equal amounts of solubilized cell protein were subjected to immunoblotting using specific antibodies to determine IR phosphorylation at Y⁹⁷² and protein content (A), IRS1/2 phosphorylation at Y⁶¹² and IRS1 protein content (B), Akt phosphorylation at S⁴⁷³ and protein content (C), GSK3β phosphorylation at S⁹ and protein content (D), AS160 phosphorylation at T⁶⁴² and protein content (E) and mTOR phosphorylation at S²⁴⁴⁸ and protein content (F). β-tubulin or β-actin were used to control protein loading. Data are the mean ± SEM of three to seven independent experiments. Each experiment included two or three replicates per experimental group. Groups denoted by different letters are significantly different (P<0.05), assessed by two-way ANOVA followed by Bonferroni post-test. NS: not significant. Representative immunoblots are shown. −/−: control cells that were not incubated with GH or insulin; −/Ins: cells that were not incubated with GH but were stimulated with insulin; GH/−: cells that were incubated with GH but were not stimulated with insulin; GH/Ins: cells that were incubated with GH and stimulated with insulin.

Figure 10.. IRS1 serine phosphorylation and GLUT4 expression in primary culture of rat cardiomyocytes incubated with GH.

Cardiac myocytes were incubated with GH (1 μg/mL) for 24 h. Cells incubated without GH were processed and analysed in parallel and used as controls (−). Equal amounts of solubilized cells protein were subjected to an ELISA assay to determine IRS1 phosphorylation at S³⁰⁷ (A) or to immunoblot analysis using specific antibodies to assess GLUT4 protein content (B). β-actin was used to control protein loading. Representative immunoblots are shown. Data are the mean ± SEM of seven samples analyzed in two independent ELISA experiments (A) and of three independent experiments (B). Groups denoted by different letters are significantly different (P<0.05), assessed by Student’s t test. NS: not significant.