Nobuaki Watanabe

A primary defect in insulin receptor in a young male patient with insulin resistance.

We studied insulin binding to cells from an insulin-resistant patient, a 5-year-old boy with clinical Rabson-Mendenhall syndrome. Decreased insulin binding was observed in three different cells: erythrocytes (37.4% of normal), cultured fibroblasts (53.3% of normal), and transformed lymphocytes (9.8% of normal). Decreased insulin binding in the cultured cells suggested that the patient had a primary defect in insulin receptors. In addition, insulin binding to the transformed lymphocytes from the patient was relatively high at lower pH compared with those in normal subjects. The cultured fibroblasts from the patient showed decreased glucose incorporation at the low insulin concentration with normal maximal stimulation, and the insulin dose response curve was shifted to the right. These results suggested that the defect resided in the receptor binding but not in the postreceptor steps. This was one of the rare cases showing decreased insulin binding clearly demonstrated in three different cells from a young male patient with extreme insulin resistance.

Effect of duration of diabetic state on insulin action in isolated rat soleus muscles.

We studied the effect of the duration of diabetic state on insulin action in skeletal muscle by measuring insulin binding, 2-deoxyglucose uptake, and intracellular glucose metabolism in isolated soleus muscles from streptozotocin-induced diabetic rats. Insulin binding to soleus muscles from diabetic rats was increased over that from controls. Glucose transport activity was determined by measuring the 2-deoxyglucose uptake at the concentration of 1 mmol/L at 25 degrees C. In the rats with diabetes of one week duration, insulin-stimulated 2-deoxyglucose uptake was not impaired, whereas basal 2-deoxyglucose uptake was decreased. However, the diabetic rats with two weeks duration revealed a 35.6% decrease in the insulin-stimulated 2-deoxyglucose uptake. Furthermore, four week duration of diabetic state led to a 60% decrease both in basal and insulin-stimulated 2-deoxyglucose uptake. Total glucose utilization was estimated as the total amount of glucose incorporated into muscle and lactate released into the medium following incubation at 37 degrees C, with 5 mmol/L glucose. The diabetic rats with one week duration did not demonstrate any changes in total glucose utilization both in basal and insulin-stimulated state. However more than two weeks duration of diabetes led to a 30% to 35% decrease both in basal and insulin-stimulated total glucose utilization, similar to the findings in the 2-deoxyglucose uptake study. We concluded that prolonged insulinopenia led to decreased glucose transport and intracellular glucose metabolism and resulted in insulin resistance in skeletal muscles.

Receptor binding and biological activity of [SerB24]-insulin, an abnormal mutant insulin

[SerB24]-insulin, the second structurally abnormal mutant insulin, and [SerB25]-insulin were semisynthesized and were studied for receptor binding and biological activity. Receptor binding and biological activity determined by its ability to increase 2-deoxy-glucose uptake in rat adipocytes were 0.7-3% of native insulin for [SerB24]-insulin and 3-8% for [SerB25]-insulin. Negative cooperative effect of these analogues was also markedly decreased. Immunoreactivity of [SerB24]-insulin was decreased whereas that of [SerB25]-insulin was normal. Markedly decreased receptor binding of [SerB24]-insulin appeared to be due to substitution of hydrophobic amino acid, Phe, with a polar amino acid, Ser, at B24.

Long-term in vitro effects of insulin on insulin binding and glucose transport

The long-term in vitro effects of insulin on insulin binding and glucose transport were studied using rat adipocytes in a time-dependent manner. Isolated fat cells were incubated with insulin (100 ng/ml) for 4, 8 and 24 h in a TCM 199 medium, at 37 degrees C, and then insulin binding (37 degrees C, 60 min) and 3-O-methylglucose transport (37 degrees C, 2 s) were determined. Decreased insulin binding was demonstrated in the cells incubated with insulin for 8 h, and Scatchard analysis revealed that receptor number was decreased to 61.7% of that of control cells. Thus, insulin-induced down regulation of receptors was evident after 8 h incubation with insulin. On the other hand, 8 h incubation with insulin resulted in markedly increased basal (i.e., in the absence of insulin) glucose transport up to 246% of control values. In the cells incubated with insulin for 24 h, maximally insulin-stimulated glucose transport was significantly increased up to 248% of control value. Thus, these results suggested that insulin-induced down regulation of receptors appeared to be coupled with increased cell-surface glucose transporters, and that there was a time-lag between down regulation of insulin receptors and increase of available glucose transporters in the plasma membrane.

Insulin binding to differentiating muscle cell line L6

We studied insulin binding to cultured differentiating muscle cell line L6. Insulin binding to the cells reached a plateau after incubation with 125I-insulin for 4 h at 22 degrees C, and was at an optimum at pH 7.8. Preincubation with 10 microM of hydrocortisone for 36 h at 37 degrees C resulted in significantly increased insulin binding (1.73 +/- 0.12 ng/mg protein for treated cells vs. 1.13 +/- 0.025 ng/mg protein for control cells, mean +/- SD, P less than 0.001). Preincubation with 1 microM of hydrocortisone or 1 microM of dexamethasone also led to increased binding. The number of insulin-binding sites per cell increased 2.5-fold in glucocorticoid-treated cells (9.7 X 10(3) sites/cell for treated vs. 3.8 X 10(3) sites/cell for control cells). Preincubation with trifluoperazine (5 microM), a calmodulin inhibitor, did not affect insulin binding to the cells. These results indicate that glucocorticoid might have some important role in regulating the number of insulin receptors in L6 muscle cells.

Inhibition of down regulation by chloroquine in cultured lymphocytes (RPMI-1788 line)

We studied the effect of chronic exposure to insulin on insulin receptors of cultured lymphocytes (RPMI-1788 line). The cells treated with insulin (2 micrograms/ml) at 37 degrees C for 12 h, showed a 36.5% decrease in the number of binding sites. Solubilized extract from the cells treated with insulin showed a 35.9% decrease of binding capacity, suggesting that insulin exposure induced the loss of total (cell surface and intracellular) receptors. Insulin-induced loss of receptors was blocked by chloroquine, suggesting that receptor loss is mediated by a chloroquine sensitive pathway. Bacitracin, which inhibited the insulin degradation, had no effect on insulin-induced receptor loss in this cell line. We also found that vitamin K5, one of the insulin mimickers, induced a 31.5% loss of insulin receptors. Therefore, the post-receptor process appeared to mediate down regulation. In cultured lymphocytes, insulin exposure caused a significant loss of total receptors, suggesting that insulin-induced receptor loss may be due to receptor degradation. Insulin-induced receptor loss is mediated by a chloroquine sensitive pathway, and is related to the post-binding process stimulated by vitamin K5.

Receptor-binding kinetics of A-14 and A-19 125I-labelled insulin

Receptor-binding kinetics and degradation of tyrosine A-14 and A-19 125I-labelled insulin was studied using cultured human lymphocytes. Receptor-binding ability of A-14 insulin was 1.5-times as high as that of A-19 insulin. Dissociation from receptors on lymphocytes showed no difference between these two labelled insulins. In association studies percent bound of A-14 insulin was 1.5-times as high as that of A-19 insulin at any time after incubation. These results suggested that lower binding affinity of A-19 insulin was due to decreased association rate, but not due to increased dissociation rate. Degradation of A-14 insulin by incubation media of lymphocytes was also 1.5-times as high as that of A-19 insulin.