Hepatic insulin resistance and inflammatory cytokine production contribute to the manifestation of the metabolic syndrome. As amino acids have been implicated in modulating insulin signaling and inflammation, we have investigated the effects of glutamine, leucine and proline on markers of inflammation and insulin sensitivity in HepG2 liver cells. Cells were incubated with IL-1beta (5 ng/mL) to stimulate IL-8 production. After 24 h, glutamine inhibited IL-8 production significantly (p<0.05) at 2, 5 and 10 mM (to 82, 73 and 72% of control), whereas leucine reduced IL-8 production significantly only at 10 mM (66%) and proline at 5 and 10 mM (71 and 52%). Glutamine, leucine and proline all reduced NF-kappaB activity after 3 h of IL-1beta stimulation at 2, 5 and 10 mM (p<0.001). Insulin-induced (1 nM) Akt phosphorylation was reduced in cells treated with tumour necrosis factor-alpha (10 ng/mL) for 24 h, but was partly restored by simultaneous incubation with glutamine, leucine and proline (25 mM). Phosphorylation of glycogen synthase kinase-3beta was unaffected by insulin stimulation and amino acid treatment. Our results indicate that glutamine, leucine and proline attenuate IL-8 production, probably through inhibition of NF-kappaB, and that they increase Akt phosphorylation in HepG2 cells.