Deletion of Asn281 in the alpha-subunit of the human insulin receptor causes constitutive activation of the receptor and insulin desensitization.

We studied the structure and function of the insulin receptor (IR) in two sisters with leprechaunism. The patients had inherited alterations in the IR gene and were compound heterozygotes. Their paternal IR allele carried a major deletion, including exons 10-13, which shifted the reading frame and introduced a premature chain termination codon in the IR sequence. This allele was expressed at a very low level in cultured fibroblasts (< 10% of total IR messenger ribonucleic acid content) and encoded a truncated protein lacking transmembrane and tyrosine kinase domains. The maternal IR allele was deleted of 3 bp in exon 3, causing the loss of Asn281 in the alpha-subunit. This allele generated levels of IR messenger ribonucleic acid and cell surface receptors similar to those seen in control fibroblasts. However, IRs from patients' cells had impaired insulin binding and exhibited in vivo and in vitro constitutive activation of autophosphorylation and tyrosine kinase activity. As a result of this IR-preactivated state, the cells were desensitized to insulin stimulation of glycogen and DNA syntheses. These findings strongly suggest that Asn281 of the IR alpha-subunit plays a critical role in the inhibitory constraint exerted by the extracellular alpha-subunit over the intracellular kinase activity.

Lipoatrophic diabetes: genetic exclusion of the insulin receptor gene.

Lipoatrophic diabetes (LD) is a syndrome with congenital or delayed onset, characterized by severe insulin resistance and generalized lipoatrophy. Using denaturing gradient gel electrophoresis and sequencing, we have investigated the contribution of defects in the insulin receptor (IR) gene in LD. First, we performed an association study between the IR gene and congenital lipoatrophy in two families with consanguineous parents and one or two affected children (patients D1, D2, and D3). Segregation analysis of intragenic polymorphisms excluded a linkage between the IR locus and the LD phenotype in both families. Second, we screened for mutations in all exons and splice site junctions of the IR gene from patients D1-D3 and 11 additional unrelated patients with congenital or delayed forms of LD. The IR sequence proved to be normal in all 14 subjects because nucleotide variations that we detected were silent. The relative levels of expression of the 2 alleles of the IR gene were evaluated by allele-specific oligonucleotide hybridization in cells from most of these patients, and no gross alteration was detected. Overall, these results provide the first clear evidence against the involvement of the IR gene in the pathogenesis of any clinical form of LD.

Increased insulin action in cultured hepatocytes from rats with diabetes induced by neonatal streptozotocin.

Previous studies have shown that Wistar rats injected at birth (n0) with STZ (n0-STZ) develop as adults a noninsulin-dependent diabetic state characterized by a lack of insulin response to glucose in vivo, a mild basal hyperglycemia, and an impaired glucose tolerance. Our former in vivo studies using the insulin-glucose clamp technique revealed an increased insulin action upon hepatic glucose production in these animals. We have now cultured hepatocytes from these mildly diabetic rats in parallel with hepatocytes from control rats, to examine more closely basal and insulin-regulated glucose production and glucose incorporation into glycogen. In addition, we extended our investigation to other hepatic functions such as lipid synthesis and amino acid transport, which could not be studied in vivo. Although glucose production from glycogenolysis or gluconeogenesis in absence or presence of glucagon was identical in the two cell populations, glucagon-stimulated glycogenolysis was more sensitive to insulin action in diabetic hepatocytes. Similarly, insulin action on glucose incorporation into glycogen, lipogenesis, and amino acid transport were enhanced in diabetic hepatocytes. The hormone effect was manifested by an increase in the sensitivity and/or in the responsiveness, reflecting the multiplicity of the pathways whereby the insulin signal is transduced through the insulin receptor to multiple postreceptor sites. To gain insight into the possible mechanism of these disturbances, we evaluated the initial insulin receptor interaction and the kinase activity of the receptor beta-subunit. In accordance with our previous study on intact livers, we found no alteration in either of these parameters in n0-STZ rat hepatocytes. Thus, the present study clearly demonstrates that these diabetic rats exhibit a postreceptor hyperresponsiveness to insulin at the cellular level. It strengthens the notion that a beta-cell deficiency with glucose intolerance does not necessarily lead to a hepatic insulin resistance.

Dual effect of metformin in cultured rat hepatocytes: potentiation of insulin action and prevention of insulin-induced resistance.

The ability of the biguanide hypoglycemic agent metformin to improve the acute effects of insulin on glucose and/or lipid metabolism was investigated in both insulin-responsive and insulin-resistant cultured rat hepatocytes: (1) metformin (20 micrograms/mL, 16 hours) increased the insulin-dependent stimulation of glycogen and lipid synthesis through an exclusive enhancement of the responsiveness without modification of the cell sensitivity to the hormone; (2) metformin neither altered basal glycogenesis from [U-14C]glucose and basal lipogenesis from [1-14C]acetate nor insulin binding. These results indicate the ability of this drug to selectively potentiate the acute action of insulin at a postreceptor step in normal liver cells. A prolonged incubation with insulin (16 hours, 5 x 10(-7) mol/L) led the hepatocytes to a state of resistance evidenced by a 50% decrease in their maximal responsiveness and sensitivity to a subsequent acute stimulation by the hormone, as assessed on lipogenesis. Addition of metformin (20 micrograms/mL) during the overnight incubation of hepatocytes with insulin prevented the decrease in cell responsiveness and sensitivity to the hormone for the stimulation of lipogenesis, thus showing that metformin was able to hamper the development of the resistant state to the hormone in this pathway. These results strongly suggest that metformin improves type 2 diabetes through an effect at the hepatic level on both insulin action and insulin-induced resistance.

In vitro studies of insulin resistance in patients with lipoatrophic diabetes. Evidence for heterogeneous postbinding defects.

We studied the binding and action of insulin in cultured fibroblasts from six patients with lipoatrophic diabetes and marked in vivo insulin resistance and from seven control subjects. The binding of insulin was not altered, which corresponds well with studies with circulating erythrocytes. Similarly, the action of the hormone on amino acid uptake (estimated by active transport of aminoisobutyric acid) was comparable in patient and control cells. Conversely, studies concerning the effect of insulin on glucose transport (estimated by facilitated diffusion of 2-deoxyglucose) or glycogen synthesis (estimated by incorporation of glucose into cellular glycogen) revealed the presence of heterogeneous alterations among the different patient cell lines. However, although the nature of the defect(s) varied among the patients, alterations in glucose metabolism were present in all cases. These data suggest the presence of primary postbinding defects in glucose cellular pathways that give rise to insulin resistance in cells from lipoatrophic diabetic patients.

Degradation of insulin receptors by hepatoma cells: insulin-induced down-regulation results from an increase in the rate of basal receptor degradation.

The degradation of insulin receptors was studied in cultured Zajdela hepatoma cells (ZHC). Receptor distribution within the cell was evaluated by estimating: i) surface receptor level on entire cells, ii) total cell receptors solubilized by Triton from cell membranes and iii) intracellular receptors solubilized from cells whose surface receptors had been inactivated with trypsin. In the absence of insulin, 80-90% of the insulin binding sites were located on the cell surface. When insulin was added, a rapid decrease of surface receptors was observed. After 2 h, their level was reduced nearly by half; this reduction was accounted for by an actual receptor loss from the cell without an increase in the intracellular pool. These results indicate that insulin enhanced the rate of receptor degradation within the cell. Basal receptor inactivation was studied by using tunicamycin which inhibits new receptor synthesis. The surface receptor number was decreased with a half-life of 7 h, while the level of internal sites remained unchanged. Both basal and insulin-activated receptor degradation were markedly slowed down by chloroquine or dansylcadaverine, indicating the importance of endocytic pathways in this process. Similarly, when de novo protein glycosylation was inhibited for 24 h by tunicamycin, both basal and insulin-activated receptor inactivation were precluded.(ABSTRACT TRUNCATED AT 250 WORDS)