Background & Aim: Metabolic syndrome (MetS) triggers abnormal insulin signaling and insulin resistance, which is associated with impaired nitric oxide (NO) availability. Endothelial NO synthase (eNOS) has been link to insulin signaling but the role of neuronal NOS (nNOS) remains unknown. Recently, nNOS is shown to be up-regulated in hypertensive left ventricular (LV) myocardium (HP). Here, we tested whether enriching metabolic substrates in vitro affects insulin regulation of LV myocyte contraction and the functional contribution of nNOS in normal and in HP rat LV myocytes. Materials & Methods: LV myocytes were isolated using enzymatic dispersion technique from normal and angiotension II-induced hypertensive rats (via osmotic minipump, 125 ng/min/kg, 10-12 weeks, males). Myocyte contraction was assessed by using a video-sarcomere detection system (IonOptix Corp, field stimulation at 2 Hz, 36 ± 1°C). Metabolic substrates (oleic acid 200 μM, palmitic acid 100μM, linolic acid 100μM, lactate 1mM, pyruvate 100μM and carnitine 50μM) were supplemented (termed nutrition full, NF solution) to the normal tyrode (NT) solution. Insulin (1-10 nM), isoprenaline (ISO, 10-50 nM), S-methyl-L-thiocitrulline (SMTC, 100 nM), L-NG-nitroarginine methyl ester (L-NAME, 1 mM) and n-acetyl cysteine (NAC, 1 mM) were used in the study. Results: Our results demonstrated that sarcomere shortenings were greater with NF in both normal and HP under basal conditions (P<0.0001, n=19 in normal and P<0.0001, n=8 in HP) and after ISO treatment (P=0.0003, n=14, n=24 in normal and P<0.0001 n=15, n=8 in HP). NAC did not affect NF increases in myocyte contraction in normal (P=0.003, n=6) and in HP (P=0.02, n=5). In normal rats, insulin pre-treatment abolished ISO-stimulation of LV myocyte contraction with NT (P=0.8, n=27) but not with NF (P<0.001, n=20), suggesting impaired insulin response in NF. L-NAME, but not SMTC, prevented the anti-adrenergic effect of insulin in these rats (L-NAME: P=0.004, n=6; SMTC: P=0.16, n=9). In contrast, ISO failed to increase LV myocyte contraction with NT in HP (P=0.1, n=15); SMTC restored the positive inotropic effect of ISO (P=0.004, n=6). The inotropic response of ISO in HP was not affected by insulin (P=0.3, n=11 in NT; P<0.0001, n=11 in NF). SMTC or L-NAME did not affect myocyte contraction the effect of ISO with NF in HP (P=0.8, n=11, n=7 with SMTC; P=0.3, n=11, n=5 with L-NAME). Conclusion: Our results show that eNOS mediates anti-adrenergic effect of insulin in normal rat LV myocytes and nNOS exerts such an effect in HP. In vitro enrichment of metabolic substrates diminished insulin responsiveness in LV myocytes by attenuating the role of NOS. Downstream mechanisms mediating impaired insulin signaling in cardiac myocytes in metabolic syndrome and in HP warrant further study.