Metabolic syndrome diminishes insulin-induced Akt activation and causes a redistribution of Akt-interacting proteins in cardiomyocytes

Abstract

Metabolic syndrome (MetS) is a cluster of cardiometabolic risk factors, with insulin resistance as a critical component for its development. Insulin signaling in the heart leads to Akt (also known as PKB) activation, a serine/threonine protein kinase, which regulates cardiac glucose metabolism and growth. Cardiac metabolic inflexibility, characterized by impaired insulin-induced glucose uptake and oxidation, has been reported as an early and consistent change in the heart of different models of MetS and diabetes; however, the evaluation of Akt activation has yielded variable results. Here we report in cardiomyocytes of MetS rats, diminished insulin-induced glucose uptake and Akt activation, evaluated by its impaired mobilization towards the plasma membrane and phosphorylation, and reflected in a re-distribution of its interacting proteins, assessed by label-free mass spectrometry (data are available via ProteomeXchange with identifier PXD013260). We report 45 proteins with diminished abundance in Akt complex of MetS cardiomyocytes, mainly represented by energy metabolism-related proteins, and also, 31 Akt-interacting proteins with increased abundance, which were mainly related to contraction, endoplasmic reticulum stress, and Akt negative regulation. These results emphasize the relevance of Akt in the regulation of energy metabolism in the heart and highlight Akt-interacting proteins that could be involved in the detrimental effects of MetS in the heart.

Fig 1. Glucose tolerance test.

A) Glucose determinations performed in control and MetS rats at 15, 30, 60 and 120 minutes after intraperitoneal injection of glucose (2g / kg). N = 6, for both groups. **P < 0.01, ***P < 0.001 vs basal (0 min). ^&&P <0.01, ^&&&P < 0.001 vs control rats. B) Area under the curve of the glucose tolerance test. ***P < 0.001 vs control rats.

Fig 2. ³H-2-deoxy-D-glucose uptake.

Cardiomyocytes of A) Control and B) MetS rats were stimulated with 100 nM insulin for 30 min, and the ³H-2-deoxy-D-glucose uptake was measured in a scintillation counter. N = 5, for both groups. *P < 0.05.

Fig 3. Akt mobilization towards caveolin-3 regions in response to insulin.

A) Control and B) MetS cardiomyocytes were stimulated with 100 nM insulin for 2 min, and the Akt (green) and caveolin-3 (red) localization was evaluated by immunostaining using confocal microscopy. C) Akt/caveolin-3 weighted colocalization coefficient. Scale bar = 20 μm. N = 3 rats, n = 30 cardiomyocytes, for both groups. ***P < 0.001.

Fig 4. Akt phosphorylation in response to insulin.

Control and MetS cardiomyocytes were stimulated with the indicated insulin concentrations for 10 min, and Akt phosphorylation in its main activation residues was evaluated by Western blot. A) pAkt Ser473. Data were normalized with the maximum value being 1 for both curves (N = 5 control, 3 MetS. *P < 0.05, ***P < 0.001 vs basal in control cardiomyocytes. ^#P < 0.05, ^##P < 0.01 vs basal in MetS cardiomyocytes); B) pAkt Thr308 (N = 3, for both groups. **P < 0.01 vs basal. ^&&P < 0.01 vs control rat cardiomyocytes).

Fig 5. Proteins differentially interacting with Akt in MetS cardiomyocytes.

Control and MetS cardiomyocytes were stimulated with 100 nM insulin for 10 min; Akt CoIP was performed, and the relative abundance of proteins interacting with Akt was assessed by label-free mass spectrometry. A) Volcano plot representing all filtered proteins: gray circles correspond to the proteins that are in complex with Akt but did not change their abundance between the MetS and control samples (69), red circles correspond to the proteins with increased abundance in Akt complex in MetS (29), green circles correspond to the proteins with diminished abundance in Akt complex in MetS (44). The X-axis corresponds to the P-value of each detected protein in the technical triplicate (values represented as -Log10), and Y-axis corresponds to the ratio of the average Hi3 intensities (MetS/C) of the technical triplicate for each detected protein (values are represented as Log2). Proteins were classified according to the biological process related to their function using STRING based on the GO database, and pie charts depicting this functional classification were constructed for B) proteins with diminished interaction with Akt in MetS and C) proteins with increased interaction with Akt in MetS.

Fig 6. Validation of a subset of Akt-interacting proteins.

Akt interaction with A) GAPDH, B) 14-3-3 ζ, and C) Hsp60 was evaluated in insulin-stimulated control and MetS cardiomyocytes (100 nM insulin for 10 min) by Akt immunoprecipitation and Western Blot. Representative image of three independent experiments. IgG heavy chain (HC) is presented as loading control. *P < 0.05, **P < 0.01 vs control. Expression levels of D) GAPDH, E) 14-3-3 ζ, and F) Hsp60 were evaluated in cardiomyocytes lysates by Western Blot. Representative image of four independent experiments. **P < 0.01 vs control.