In-depth phosphoproteomic profiling of the insulin signaling response in heart tissue and cardiomyocytes unveils canonical and specialized regulation.

BACKGROUND: Insulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent. METHODS: Insulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements. RESULTS: We quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (InsrY1351, Y1175, Y1179, Y1180) itself as well as the Insulin receptor substrate protein 1 (Irs1S522, S526). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized cardiomyocyte proteins, such as Kcnj11Y12 and DspS2597. Details of all phosphorylation sites are provided. CONCLUSION: We present a first global outline of the insulin-induced phosphorylation signaling response in heart tissue and in isolated adult cardiomyocytes, detailing the specific residues with changed phosphorylation abundances. Our study marks an important step towards understanding the role of insulin signaling in cardiac diseases linked to insulin resistance.

Effects of Roux-en-Y gastric bypass on circulating follistatin, activin A, and peripheral ActRIIB signaling in humans with obesity and type 2 diabetes.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) surgery is a therapeutic intervention for morbid obesity and type 2 diabetes (T2D) that improves metabolic regulation. Follistatin (Fst) could be implicated in improved glycemia as it is highly regulated by RYGB. However, it is unknown if metabolic status, such as T2D, alters the Fst response to RYGB. In addition, the effect of RYGB on the Fst target, activin A, is unknown in individuals with obesity and T2D, but is needed to interpret the functional effects of altering Fst. Finally, whether Fst-regulated intracellular signaling contributes to beneficial effects of RYGB is undetermined. METHODS: Circulating Fst and activin A were measured before, 1 week, and 1 year after RYGB surgery in a total of 20 individuals with obesity, 10 with normoglycemia (NGT) and 10 with preoperative T2D. Intracellular signaling downstream of the Activin receptor type IIB (ActRIIB) signaling pathway was analyzed in skeletal muscle and adipose tissue. RESULTS: The doubling in circulating Fst observed in subjects with NGT 1-week and 1-year post surgery was absent in T2D. After 1 week, RYGB reduced activin A by 27% (p < 0.001) and 20% (p < 0.01) in subjects with NGT and T2D, respectively; a reduction that tended to be maintained in the subjects with T2D at 1-year post-RYGB (-15%; p = 0.0592). RYGB had no effects on skeletal muscle ActRIIB signaling. In contrast, adipose tissue phosphorylation of SMAD2Ser465/467, p70S6KThr389, S6RPSer235/236, and 4E-BP1Thr37/49 was highly regulated, particularly 1-year post-RYGB (p < 0.05). CONCLUSIONS: In subjects with preoperative T2D, RYGB did not increase circulating Fst contrasting subjects with NGT, while the reduction in activin A was maintained. ActRIIB signaling was upregulated in adipose tissue, but not skeletal muscle, following RYGB in both individuals with NGT and T2D. Our results suggest a role of adipose tissue ActRIIB signaling for the beneficial effects of RYGB surgery.