Effect of thyroid hormone binding proteins on insulin receptor binding of B1-thyronine-insulin analogues

Abstract

Certain thyronine-insulin analogues, which form non-covalent complexes with plasma proteins, have been shown to act preferentially in the liver. We hypothesized that this property may be dependent on the ability of the analogue to bind to the insulin receptor without prior dissociation from the binding protein. NaB1-L-thyroxyl-insulin, NaB1-3,3',5'-triiodothyronine-insulin, NaB1-D-thyroxyl-insulin and NaB1-L-thyroxyl-aminolauroyl-insulin were compared with insulin for their capacity to inhibit the binding of [125I]TyrA14-insulin to rat liver plasma membrane in albumin-free buffer. Effective doses at 50% maximum inhibition of binding (ED50) were calculated with and without addition of the thyroid hormone binding proteins transthyretin, thyroxine binding globulin and human serum albumin. The binding of thyronine-insulin analogues to insulin receptors was inhibited in a dose-dependent manner by the addition of thyroid hormone binding proteins at concentrations in the physiological range. Complexes of thyronine-insulin analogues with thyroid hormone binding proteins exhibit impaired insulin receptor binding affinities compared with those of the analogues in their free form. Hepatoselectivity in vivo may not depend on binding of the intact complexes to hepatocytes. These results have implications for the physiological role of hormone binding proteins and the in vivo properties of other insulin analogues which bind to plasma proteins.

Figure 1. Schematic representation of TIAs

Thyronine covalently attached by a peptide link to the ε-amino group of Phe^B1. The spacer chain in B1-L-T4-AL-Ins is an amino lauryl (12 carbon chain).

Figure 2. FPLC elution profile of analogues

FPLC elution profile of analogues [(▾) H-Ins, (○) B1-L-T4-Ins, (•) B1-rT3-Ins and (▾) B1-D-T4-Ins] after overnight incubation with TBG. A Superose 12 HR 10/30 column (Pharmacia FPLC system, Pharmacia LKB Ltd, U.K.) was equilibriated with 0.1 mol/l phosphate buffered saline (PBS) buffer, pH 7.2, and eluted with it at 0.5 ml·min⁻¹, under a column pressure of 1.2 MPa. Fractions of 0.5 ml from eluate 5.5 ml–25 ml were collected by a programmable fraction collector (Pharmacia LKB Ltd, U.K.). The column was standardized by eluting a series of marker proteins of known molecular mass. Analogue concentration in the eluate fractions was measured by RIA. Bound (fractions eluting from to 5.5 to 15 ml) (%) and unbound (fractions eluting from 15.5–25 ml) (%) components of the analogues was calculated. The proportion of each analogue bound to the THBPs was calculated from the RIA concentrations in the eluate fractions.

Figure 3. Equilibrium binding curves of the TIAs to LPM in the absence of thyroid hormone binding proteins

Inhibition of binding of [¹²⁵I]Tyr^A14 monoiodinated H-Ins to rat LPM by the analogues in KRP buffer pH 7.8, containing γ globulin (1% w/v) in the absence of THBPs. (A) (○) B1-L-T4-Ins, (•) B1-rT3-Ins and (▿) H-Ins, and (B) (▿) H-Ins, (○) B1-L-T4-Ins, (▿) B1-D-T4-Ins and (♦) B1-L-T4-AL-Ins.

Figure 4. Equilibrium binding curves of the B1-L-T4-Ins to LPM in the presence of thyroid hormone binding proteins

Inhibition of binding of [¹²⁵I]Tyr^A14 monoiodinated H-Ins to rat LPM by (○) B1-L-T4-Ins in KRP buffer pH 7.8, containing γ globulin (1% w/v) in the presence of increasing concentration of TBG [(▴) 0.023 μmol/l, (▪) 0.068 μmol/l, (♦) 0.135 μmol/l and (▾) 0.27 μmol/l] (A) and in the presence of increasing concentration of HSA [(▴) 55 μmol/l, (▪) 110 μmol/l, (♦) 275 μmol/l and (▾) 550 μmol/l] (B).

Figure 5. Equilibrium binding curves of the B1-rT3-Ins to LPM in the presence of thyroid hormone binding proteins

Inhibition of binding of [¹²⁵I]Tyr^A14 monoiodinated H-Ins to rat LPM by (○) B1-rT3-Ins in KRP buffer pH 7.8, containing γ globulin (1% w/v) in the presence of (▴) TBG (0.135 μmol/l) and (♦) TTR (4.6 μmol/l) (A) and in the presence of increasing concentration of HSA HSA [(▴) 55 μmol/l, (♦) 110 μmol/l, (▪) 275 μmol/l] (B).