The aim of the studies presented here was to discover novel insulin analogues that could restore the physiological portal-arterial insulin gradient, that is lost by subcutaneous insulin treatment. Fatty acid acylation technology has provided B29 acylated insulin analogues for clinical use, and we here present an acylation scan of 13 solvent-exposed residues of the insulin molecule (A8, A9, A14, A15, A18, A21, A22, B1, B2, B3, B22, B27, and B29) aiming at discovering analogues with high insulin receptor affinity in presence of albumin. The albumin-binding side chain was chosen to mediate high affinity for serum albumin. The A22 position stood out as the single acylation position that conferred high insulin receptor binding affinity in presence of 1.5% HSA, 11% relative to human insulin, as well as strong HSA binding affinity. The A22 acylated analogue, Insulin 327, was shown to be hepatopreferential in a mouse model, where insulin-mediated phosphorylation of hepatic insulin receptors was increased over phosphorylation of insulin receptors in muscle and fat tissues following equi-molar intravenous dosing of the A22 acylated insulin relative to that of human insulin. Additionally, insulin 327 has been shown in dogs to functionally restore the physiological portal-arterial insulin gradient leading to normalization of hepatic and non-hepatic glucose metabolism.