Lorri D. Griffin

Increased glucose transport by human fibroblasts with a heritable defect in insulin binding.

Insulin and IGF-I binding and their regulation of hexose transport were evaluated in skin fibroblasts cultured from a family (Atl) whose proband had leprechaunism, hypoglycemia, and severe insulin resistance. High affinity insulin binding to proband Atl cells was absent, and partially, but equally, impaired in fibroblasts from his related parents. IGF-I binding to his cultured fibroblasts was within the normal range. Cells from proband Atl had insulin receptor mRNAs similar to control fibroblasts. 3-O-Methyl-D-glucose (OMG) transport by proband Atl was threefold higher than in control fibroblasts (37.7 v 7.6-11 nmol/mL/s) and was insulin-insensitive. Proband Atl fibroblasts had a threefold increase in the Vmax for OMG entry and a concomitant increase in the number of D-glucose-inhibitable cytochalasin B binding sites on their plasma membrane. Similar levels of glucose transporter mRNA were observed in control and proband Atl fibroblasts. These results suggest that fibroblasts from patient Atl have a genetically transmitted mutation in the alpha subunit of their insulin receptor. In the homozygous affected proband, this mutation impairs insulin binding and causes elevated, insulin-insensitive glucose transport. The dysfunction resulting from this mutation is similar to that introduced in Chinese hamster ovary cells by transfection with a truncated alpha subunit.

Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin

The regulation of 3-O-methyl-D-glucose (OMG) uptake by insulin and phorbol esters was studied in cultured human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12-15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with no significant changes of the Km (4-6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.

Insulin-receptor autophosphorylation and kinase activity are constitutively increased in fibroblasts cultured from a patient with heritable insulin-resistance

Mutations in the insulin receptor gene have been described in families with the inherited insulin-resistant syndrome leprechaunism. At a cellular level, these mutations result in decreased insulin binding and impaired insulin stimulation of receptor autophosphorylation and sugar transport. By contrast, we previously found that fibroblasts cultured from leprechaun patient Atl had constitutively increased sugar transport, even though insulin binding was markedly reduced. Here we report that these fibroblasts have basal insulin-receptor autophosphorylation and kinase activity constitutively increased above insulin-stimulated control cells.