Pierre De Meyts

Role of kinase C in the insulin-like effects of human growth hormone in rat adipocytes

Insulin and to a smaller extent, human growth hormone (hGH), both stimulate lipogenesis in isolated rat adipocytes preincubated 4 hours in the absence of hormone. The non-additivity of maximal doses suggested that hGH may share a subset of the metabolic pathways stimulated by insulin. We explored whether kinase C may be involved in the common lipogenic effect of both hormones. The stimulation of lipogenesis by phorbol ester 12-myristate 13-acetate (PMA) (an activator of kinase C) was not additive to the stimulation by either insulin or hGH. Downregulation of kinase C resulted in a marked decrease of the maximal insulin effect (44 +/- 9%) and even more of the hGH effect (64 +/- 14%). These data suggest that kinase C either mediates part of, or modulates, the effect of insulin and hGH on lipogenesis.

Reversal of insulin-induced negative cooperativity by monoclonal antibodies that stabilize the slowly dissociating ("Ksuper") state of the insulin receptor.

Two monoclonal antibodies to the insulin receptor, MA-5 and MA-20, unlike other monoclonal antibodies, do not mimick the accelerating effect of insulin on the dissociation of 125I-insulin from the receptors (negative cooperativity). On the contrary, MA-5 and MA-20 markedly slow down the dissociation rate. We show now that MA-5 and MA-20 are potent antagonists of the negative cooperativity induced by insulin, and reverse the insulin-induced acceleration whether added simultaneously with insulin or after insulin. The reversal of the insulin-induced acceleration is almost immediate. These data strengthen the concept therefore that the insulin-receptor complex has access to alternative conformational states that can be stabilized by ligand-induced site-site interactions.

Acridine orange, an inhibitor of protein kinase C, abolishes insulin and growth hormone stimulation of lipogenesis in rat adipocytes

To determine whether protein kinase C plays a role in the actions of insulin and growth hormone in rat adipocytes, we tested the effect of acridine orange, a potent inhibitor of kinase C, on the lipogenic activity of both hormones. This compound completely inhibited the effects of insulin, growth hormone and phorbol ester 12‐myristate 13‐acetate, whereas 9‐acridine carboxylic acid, an analog of acridine orange which does not inhibit kinase C, had no effect. Acridine orange did not act through inhibition of hormone binding. These data are consistent with the involvement of kinase C in the action of insulin and growth hormone on lipogenesis in rat fat cells.

Insulin and IGF-I Receptor Structure and Binding Mechanism

The insulin and IGF-I receptors are members of the superfamily of receptor tyrosine kinases (RTKs). Unlike most RTKs that are single-chain monomeric transmembrane polypeptides, the insulin and IGF-I receptors are covalent dimers composed of two extracellular α subunits and two transmembrane β subunits containing the tyrosine kinase domains. The α subunits contain the ligand binding sites, of which at least three subdomains have been defined by photoaffinity crosslinking, alanine-scanning mutagenesis or minimized receptor constructs. All RTKs are dimeric or oligomeric in the ligand-activated form. The residues of insulin involved in receptor binding have been mapped by alanine-scanning mutagenesis. They form at least two major epitopes that partially overlap with the dimer- and hexamer-forming surfaces of the insulin molecule, and we propose that insulin is using those surfaces to asymmetrically cross-link the two receptor α subunits. This mechanism provides a structural basis for high affinity binding and negative cooperativity, and probably also operates in the IGF-receptor interaction. It also provides a structural basis for the approximation and transphosphorylation of the kinase domains and triggering of the signalling cascade.