Abnormal myocardial insulin signalling in type 2 diabetes and left-ventricular dysfunction

Abstract

Aims: Whole body and myocardial insulin resistance are features of non-insulin-dependent diabetes mellitus (NIDDM) and left-ventricular dysfunction (LVD). We determined whether abnormalities of insulin receptor substrate-1 (IRS1), IRS1-associated PI3K (IRS1-PI3K), and glucose transporter 4 (GLUT4) contribute to tissue-specific insulin resistance.

Methods and results: We collected skeletal muscle (n = 27) and myocardial biopsies (n = 24) from control patients (n = 7), patients with NIDDM (n = 9) and patients with LVD (n = 8), who were characterized by euglycaemic-hyperinsulinaemic clamp and positron emission tomography. Comparative studies were carried out in three mouse models. We demonstrate an unrecognized reduction of IRS1 in skeletal muscle of LVD patients and an unexpected increase in cardiac IRS1-PI3K activity in NIDDM and LVD patients. In NIDDM, there was a concomitant reduction in sarcolemmal GLUT4, whereas in patients with LVD sarcolemmal GLUT4 was increased. We confirm activation of IRS1-PI3K and reduction in sarcolemmal GLUT4 in the insulin resistant ob/ob mouse heart where we also demonstrate perturbation of GLUT4 docking and fusion. A direct relationship between PI3K and GLUT4 was demonstrated in mice expressing activated PI3K in the heart and increased GLUT4 at the sarcolemma was confirmed in a mouse model of LVD.

Conclusion: Our data show that the mechanisms of myocardial insulin resistance are different between NIDDM and LVD.

Figure 1

Consort diagram of human study design. NIDDM, non-insulin-dependent diabetes mellitus.

Figure 2

Whole body glucose disposal and myocardial glucose utilization. (A) Whole body glucose utilization under euglycaemic–hyperinsulinemic clamp conditions in patients with normal ventricular function without diabetes (control), patients with normal ventricular function and non-insulin-dependent diabetes mellitus (NIDDM) and patients with left-ventricular dysfunction (LVD); (**P = 0.002 vs. control, ^‡P = 0.001 vs. control, ^†P = 0.05 vs. LVD). (B) Myocardial glucose utilization under euglycaemic–hyperinsulinemic clamp conditions as determined by positron emission tomography (PET) with ¹⁸F-fluorodeoxyglucose; (*P = 0.02 vs. control, ^¶P = 0.006 vs. control).

Figure 3

Insulin receptor substrate-1 (IRS1) protein levels and activity in skeletal muscle. (A) Immunoblot of IRS1 protein levels in total cell lysates from skeletal muscle biopsies. Levels of IRS1 were unchanged in patients with non-insulin-dependent diabetes mellitus when compared with controls. (B) Representative immunoblot of phosphotyrosine- IRS1 levels in skeletal muscle extracts as determined by phosphotyrosine immunoprecipitation and insulin receptor substrate-1 immunoblotting. (C) Quantification of total IRS1 levels in skeletal muscle biopsies in patient groups in optical units. (D) Quantification of phosphotyrosine- IRS1 levels in skeletal muscle biopsies in patient groups in optical units. **P = 0.001 vs. control.

Figure 4

Activation of insulin receptor substrate-1-phosphatidylinositol-3 kinase (IRS1-PI3K) and the insulin receptor in the diabetic heart. (A) Autoradiograph of myocardial IRS1-PI3K activity in cardiac biopsies as determined by in vitro kinase assay. (B) Quantification of IRS1-PI3K activity in arbitrary optical units. Data were transformed to a logarithmic scale (base 2) for linear regression analysis; *P = 0.02 vs. control, ***P < 0.0001 vs. control. (C) Inverse log-linear association of myocardial IRS1-PI3K activity and whole body glucose utilization across the study population by multivariate regression analysis (r = −0.68, P = 0.01). (D) Positive correlation between fasting insulin levels and myocardial IRS1-PI3K activity (r = 0.64, P = 0.007). (E) Representative immunoblot of Akt kinase activity towards GSK3β in patients with non-insulin-dependent diabetes mellitus and controls. (F) Representative immunoblot of IRS1 following immunoprecipitation of the insulin receptor; IRS1 is located at 150 kDa and a non-specific protein band is seen at ∼220 kDa. (G) Autoradiograph of receptor tyrosine kinase arrays using pooled samples (n = 3–4) from the patient groups and controls. The two spots at the four corners of the arrays are internal positive controls. Highlighted in the red rectangle is the signal (two technical replicates) generated by the phosphorylated insulin receptor. The experiment was repeated with similar results.

Figure 5

Myocardial glucose transporter 4 (GLUT4) levels in patients with non-insulin-dependent diabetes mellitus or left-ventricular dysfunction. (A) Immunoblot of GLUT4 levels in total cell lysates from patients with non-insulin-dependent diabetes mellitus and controls. (B–D) Sucrose gradient fractionation of cardiac muscle proteins. (B) Upper panel, representative immunoblot of GLUT4 expression at the plasma membrane fraction (Mbn) and across sucrose gradient fractions (1–10) in a control patient. Lower panel, immunoblot of GLUT4 expression at the Mbn and across sucrose gradient fractions (1–10) in a patient with non-insulin-dependent diabetes mellitus. (C) Immunoblot of GLUT4 levels at the plasma membrane fraction (upper panel) and Caveolin-3 (Cav-3) as loading control (lower panel) in controls or patients with non-insulin-dependent diabetes mellitus. (D) Quantification of GLUT4 expression at the sarcolemma in control patients (n = 3) and patients with non-insulin-dependent diabetes mellitus (n = 3) in arbitrary optical units; (*P = 0.01). (E and F) Myocardial GLUT4 levels in patients with left-ventricular dysfunction. (E) Immunoblot of GLUT4 levels at the sarcolemma (upper panel) and Caveolin-3 as loading control (lower panel) in control patients or patients with left-ventricular dysfunction. (F) Quantification of GLUT4 expression at the sarcolemma in control patients (n = 3) and patients with left-ventricular dysfunction (n = 4) in arbitrary optical units; *P = 0.01.

Figure 6

Myocardial IRS1-PI3K, Akt, and sarcolemmal GLUT4 in ob/ob mice. (A) Autoradiograph of in vitro kinase assay of IRS1-PI3K activity in the mouse heart. (B) Quantification of IRS1-PI3K activity in arbitrary optical units; (*P < 0.01). (C) Immunoblot of the insulin receptor beta (IR-β) subunit following phosphotyrosine immunoprecipitation (upper panel) and immunoblot of insulin receptor substrate-1 following IR-β immunoprecipitation (lower panel). (D) Fasting serum insulin levels of ob/+ and ob/ob mice (**P = 0.001). (E) Immunoblot of GLUT4 levels in total heart lysates from ob/+ and ob/ob mice. (F) Immunoblots of GLUT4 in the sarcolemmal fraction (Mbn), across subcellular fractions (1–10) and in the cytosol (cy) in ob/+ controls (upper panel) and ob/ob (lower panel) mice. (G) Immunoblot of GLUT4 levels at the sarcolemma and Caveolin-3 (Cav-3) loading control. (H) Quantification of GLUT4 levels at the sarcolemma in arbitrary optical units; ob/+ (n = 4), ob/ob (n = 4), *P < 0.01. (I) Representative immunoblots of myocardial phospho-Akt (top panel), total Akt (middle panel), and GAPDH (lower panel) expression in ob/+ and ob/ob mice. (J) Representative immunoblots of myocardial phospho-Akt (top panel), total Akt (middle panel), and GAPDH (lower panel) expression in ob/+ and ob/ob mice at basal state and after 30 min of insulin stimulation. (K) Quantification of phospho-Akt to total Akt ratio in ob/+ and ob/ob with and without insulin stimulation in ratio units. *P < 0.01 vs. ob/+ basal, **P < 0.001 vs. ob/+ basal, and ***P < 0.0001 vs. ob/+ basal.

Figure 7

Glucose transporter 4 in mice with left-ventricular dysfunction or constitutively active phosphatidylinositol-3 kinase (caPI3K). (A) Representative immunoblots of GLUT4 in the sarcolemmal fraction (Mbn), across subcellular fractions (1–9) (upper panel) and of Cav-3 loading control (lower panel) in sham-operated mice. (B) Representative immunoblots of GLUT4 in the sarcolemmal fraction (Mbn), across subcellular fractions (1–9) (upper panel) and of Cav-3 loading control (lower panel) in transverse aortic constriction-induced left-ventricular dysfunction mice. The experiment was repeated three times with similar results. (C) Quantification of GLUT4 levels at the sarcolemma in arbitrary optical units; sham (n = 4) and transverse aortic constriction (n = 4). (D) Representative immunoblots of phospho-Akt, total Akt, and GAPDH (loading control) in transverse aortic constriction vs. controls (n = 3). (E) Representative immunoblots of GLUT4 in the sarcolemmal fraction (Mbn) and across subcellular fractions (1–9) in wild-type (WT, upper panel) and caPI3K (lower panel) mice. (F) Immunublot of GLUT4 levels in total cell lysates from control (WT) and caPI3K mice. (G) Quantification of GLUT4 levels at the sarcolemma in arbitrary optical units; wild-type (n = 3), caPI3K (n = 3), (*P < 0.01).

Figure 8

Expression of proteins involved in glucose transporter 4 translocation and docking in ob/ob mice. (A) Representative immunoblots of AS160, Syntaxin 4, Scamp3, SNAP23, and GAPDH (loading control) in ob/ob mice vs. controls (n = 3). (B–E) Quantification of AS160, Syntaxin 4, Scamp3, and SNAP23 expression in ob/ob mice vs. controls in ratio units normalized to GAPDH; (**P = 0.001).

Figure 9

A potential model for the interaction of glucose transporter 4 containing vesicles with the fusion/docking machinery in the mouse diabetic heart. ↑for increased expression, ↓for decreased expression, and for translocation to the membrane.