Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification

Abstract

Aims/hypothesis: Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy. We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet (HFD).

Methods: Rats were exposed to an HFD or a low-fat diet (LFD). In vivo cardiac function was monitored by echocardiography. Substrate uptake and utilisation were determined in isolated cardiomyocytes.

Results: Feeding an HFD for 8 weeks induced left ventricular dilation in the systolic phase and decreased fractional shortening and the ejection fraction. Insulin-stimulated glucose uptake and proline-rich Akt substrate 40 phosphorylation were 41% (p < 0.001) and 45% (p < 0.05) lower, respectively, in cardiomyocytes from rats on the HFD. However, long-chain fatty acid (LCFA) uptake was 1.4-fold increased (p < 0.001) and LCFA esterification into triacylglycerols and phospholipids was increased 1.4- and 1.5-fold, respectively (both p < 0.05), in cardiomyocytes from HFD compared with LFD hearts. In the presence of the CD36 inhibitor sulfo-N-succinimidyloleate, LCFA uptake and esterification were similar in LFD and HFD cardiomyocytes. In HFD hearts CD36 was relocated to the sarcolemma, and basal phosphorylation of a mediator of CD36-trafficking, i.e. protein kinase B (PKB/Akt), was increased.

Conclusions/interpretation: Feeding rats an HFD induced cardiac contractile dysfunction, which was accompanied by the relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB/Akt. The permanent presence of CD36 at the sarcolemma resulted in enhanced rates of LCFA uptake and myocardial triacylglycerol accumulation, and may contribute to the development of insulin resistance and diabetic cardiomyopathy.

Fig. 1

a Blood glucose levels after an oral glucose load in rats fed the HFD (closed circles; n = 16) and the LFD (open circles; n = 8). Data are means±SE. b Representative echocardiographic M-mode images from at least three cardiac contractile cycles at the start of the diet intervention and 8 weeks after initiation of the LFD and HFD, respectively

Fig. 2

Rates of 2-deoxyglucose uptake in cardiomyocytes from rats fed the LFD (open bars) and the HFD (filled bars). Cardiomyocytes were incubated with DMSO (Basal), insulin (INS) or oligomycin (Oli) before measurement of 2-deoxyglucose uptake. Data are expressed as wet cell mass and are means±SE; n = 8. *p < 0.02, agonist effect; #p < 0.02, diet effect

Fig. 3

Immunoblots (a, c) and quantification of PRAS40-Thr246 (b) and ACC-Ser79 (d) phosphorylation after incubation of cardiomyocytes from LFD- and HFD-fed rats with DMSO (Basal), insulin (INS) or oligomycin (Oli). Data are means±SE; n = 5. *p < 0.05, agonist effect; #p < 0.05, diet effect

Fig. 4

Rates of palmitate uptake (a), oxidation (b) and esterification into intracellular triacylglycerols and phospholipids (c) in cardiomyocytes from LFD-fed rats (open bars) and HFD-fed rats (filled bars). Cardiomyocytes were incubated with DMSO (Basal), insulin (INS), oligomycin (Oli) or SSO before measurement of palmitate uptake. Data are wet cell mass and are means±SE; n = 8. *p < 0.05, agonist effect; #p < 0.05 diet effect

Fig. 5

Expression and subcellular localisation of CD36. Expression of CD36 in ventricular lysates of HFD and LFD rats. Ponceau S staining confirmed that the filters contained equal amounts of ventricular extracts (a). Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from rats fed an LFD (b, d) or HFD (c, e) for 8 weeks. Rats received an i.p. injection of saline (b, c) or insulin (d, e) 30 min before killing. Photographs are representative of two independent experiments performed on three rats per experimental group. Arrows indicate intercalated discs. Scale bar indicates 25 μm

Fig. 6

Effect of the duration of HFD feeding on CD36 localisation and phosphorylation of PKB/Akt. Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from saline-injected rats fed an LFD (a) or HFD (b) for 4 weeks. Photographs are representative of two or three independent experiments performed on six rats per experimental group. The arrows indicate intercalated discs. The scale bar indicates 25 μm. c Immunoblot and quantification d of PKB/Akt-Ser473 phosphorylation levels in ventricular homogenates from saline-injected rats fed an LFD (open bars) or HFD (filled bars) for 4 and 8 weeks, respectively. Data are means±SE, n = 8. *p < 0.05, diet effect