Inhibitory effects of cerulenin on protein palmitoylation and insulin internalization in rat adipocytes.

Protein acylation by long-chain fatty acids has been suggested as a necessary step in membrane trafficking. Because several insulin effects are dependent upon membrane trafficking, the cellular effects of the protein acylation inhibitor cerulenin were examined. Cerulenin blocked palmitoylation of selected rat adipocyte proteins including CD36, the dominant marker for palmitoylation in adipocytes. To measure cerulenin's effects on insulin internalization, rat adipocytes were incubated with 125I-insulin at 37 degrees C in the presence or absence of cerulenin. Surface-bound and intracellular insulin were discriminated by the sensitivity of the former to rapid dissociation by a pH 3 buffer at 4 degrees C. Insulin internalization was inhibited 85% by 0.3 mM cerulenin. Inhibition required preincubation with the agent, was irreversible, was not dependent upon protein synthesis, and was not the result of ATP depletion. Cerulenin was also found to inhibit insulin-stimulated glucose uptake and acetyl-CoA carboxylase activity. Cerulenin had no effect on basal glucose uptake and utilization or on the uptake and retention of fatty acids. In summary, protein acylation may be an important step in insulin-regulated cellular functions dependent upon membrane trafficking, such as insulin internalization.

Divergence between GLUT4 mRNA and protein abundance in skeletal muscle of insulin resistant rats.

Hyperglycemia and skeletal muscle insulin resistance coexist in uncontrolled type 2 diabetes mellitus. Similar defects in insulin action were observed in glucose-infused, normal rats, a model of glucose toxicity. In these rats insulin-stimulated glucose uptake by skeletal muscle was decreased due to a post-receptor defect. We investigated whether the impaired glucose uptake resulted from a decrease in the abundance of the predominant muscle glucose transporter (GLUT4) mRNA and/or protein. GLUT4 protein abundance in the hyperglycemic rats was not different from the control group despite a 50% decrease in muscle glucose uptake. GLUT4 mRNA abundance was 2.5-fold greater in the hyperglycemic rats as compared to the control animals. We conclude that the coexistence of hyperglycemia and hyperinsulinemia results in (1) a defect in GLUT4 compartmentalization and/or functional activity and (2) a divergence between GLUT4 mRNA levels and translation.

Insulin resistance in normal rats infused with glucose for 72 h.

Insulin resistance is accentuated during periods of poor metabolic control in human non-insulin-dependent diabetes mellitus. The role of hyperglycemia in this suppression of insulin action is not clear. If glucose impairs insulin action, then the effect should be reproducible in vivo in tissues of normal intact rats. To test this possibility, normal rats were continuously administered 50% glucose in water (60-66 mg.kg-1.min-1) via an indwelling jugular catheter. After 72 h, these animals were hyperglycemic, hyperinsulinemic, and glucosuric compared with control rats infused for 72 h with normal saline (P less than 0.01). Basal glucose uptake in vivo was greater in muscle of glucose-infused rats. Insulin-stimulated glucose uptake in vivo and in vitro (by perfused hindquarters and isolated adipocytes) were suppressed in the glucose-infused group (P less than 0.01). Glycogen synthase activity was reduced 40% in extracts of muscle and adipose tissue of hyperglycemic rats. Basal and isoproterenol-stimulated lipolysis were increased, whereas insulin suppression of lipolysis was blunted in adipocytes from glucose-infused animals (P less than 0.01). Glucose infusion did not alter insulin binding by isolated adipocytes or solubilized skeletal muscle insulin receptors. These results suggest that a 72-h in vivo glucose infusion impaired insulin action in muscle and adipose tissue of normal rats by inducing postbinding defects similar to those observed in human diabetes mellitus during intervals of deteriorated metabolic control.

Cushing's syndrome in pregnancy.

Fertility and childbearing rarely occur in Cushing's syndrome because amenorrhea, oligomenorrhea, infertility, and abortions characterize the disease. Currently, a total of 53 cases of Cushing's syndrome and pregnancy have been reported. When Cushing's syndrome occurs during pregnancy, approximately 56 per cent of the cases are associated with adrenal cortical adenoma or carcinoma. Excluding Cushing's disease, nearly 21 percent of the cases are caused by adrenal carcinoma. The maternal catabolic state of glucocorticoid excess contributes to poor fetal outcome with many of the cases complicated by either fetal wastage or prematurity. However, congenital malformations are not seen more frequently than in normal pregnancy. Pregnancy may or may not influence Cushing's syndrome, but Cushing's syndrome definitely complicates pregnancy.

Relationships between cell surface insulin binding and endocytosis in adipocytes.

Chymotrypsin substrate analogues, such as N-acetyl-Tyr ethyl ester, have recently been demonstrated to inhibit the endocytic uptake of insulin in isolated rat adipocytes. In this study, the effects of N-acetyl-Tyr ethyl ester on cell surface insulin binding and dissociation were examined. Surface-bound 125I-insulin was distinguished from intracellular 125I-insulin by the sensitivity of the former to rapid dissociation with an acidic buffer (pH 3.0). Plateau levels of surface-bound insulin at 37 degrees C were increased 70% by inhibiting the internalization pathway. This increase was temperature and insulin concentration dependent. Thus differences in surface binding were small at 12 degrees C and also at high (100-200 ng/ml) insulin concentrations. Inhibition of internalization with N-acetyl-Tyr ethyl ester markedly slowed the loss of surface-bound insulin observed during dissociation studies. After 20-30 min of dissociation, the remaining levels of surface-bound insulin were three- to fourfold higher in treated adipocytes compared with control adipocytes. Added unlabeled insulin retained its ability to accelerate the dissociation of insulin in N-acetyl-Tyr ethyl ester-treated cells. These observations indicate that the internalization pathway is a quantitatively important factor in determining levels of surface binding at 37 degrees C and in determining the rate of deactivation of insulin binding.

Insulin-stimulated glucose transport and insulin internalization share a common postbinding step in adipocytes.

We recently demonstrated that chymotrypsin substrate analogues inhibit receptor-mediated insulin internalization in isolated rat adipocytes. In this study, the effect on glucose transport of inhibiting insulin internalization with these agents was examined. Glucose transport was assayed by measuring [3H]-2-deoxyglucose uptake, and internalized insulin was measured after rapidly dissociating surface-bound insulin with an acidic buffer. The chymotrypsin substrate analogue N-acetyl-Tyr ethyl ester inhibited insulin internalization by 85% while increasing surface-bound insulin by 80-110%. Under these conditions, ATP levels were minimally altered, and basal glucose transport was unchanged; however, insulin-stimulated glucose transport was decreased by 86%. The inhibition of insulin-stimulated glucose transport was not overcome by supramaximal concentrations (400 ng/ml) of insulin. When insulin internalization and insulin-stimulated glucose transport were measured in the presence of increasing concentrations of N-acetyl-Tyr ethyl ester (0.1-1 mM), a strong and highly significant correlation (r = .97, P less than .001) was found between inhibition of insulin internalization and inhibition of insulin-stimulated glucose uptake. Fragments of N-acetyl-Tyr ethyl ester that do not inhibit insulin internalization were also without effect on insulin-stimulated glucose transport. In addition to N-acetyl-Tyr ethyl ester, four other chymotrypsin substrate analogues that are effective inhibitors of insulin internalization also markedly inhibited insulin-stimulated glucose transport. These results indicate that insulin internalization and insulin-stimulated glucose transport share a common postbinding step in adipocytes and that this step is inhibitable by chymotrypsin substrate analogues.

Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes.

To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.

Insulin internalization and degradation in adipocytes from normal and type II diabetic subjects.

We studied the ability of isolated adipocytes from normal and type II diabetic subjects to internalize and process [125I]insulin. Adipocytes were incubated with [125I]insulin at 16 or 37 C, and at various times total cell-associated, surface-bound, and intracellular insulin were quantitated using an acid-barbital extraction technique which quickly removes cell surface insulin, leaving behind the intracellular insulin. Insulin internalization was slow in normal adipocytes at 16 C, such that only 13% of total cell-associated insulin was intracellular after 2 h of incubation. In contrast, internalization was rapid at 37 C, such that the intracellular pool of insulin was near maximal by 30 min and accounted for approximately 40% of the total cell-associated insulin. Sephadex G-50 column chromatography of the intracellular insulin demonstrated that more than 95% of this pool coeluted with native insulin. In adipocytes from the diabetic subjects, approximately 45% of total cell-associated insulin was intracellular after 30 min of incubation at 37 C. After 60 min of incubation at 37 C, the percentages of total cell-associated and surface-bound insulin were significantly lower in adipocytes from diabetic compared to normal subjects [1.81 +/- 0.31% (+/- SEM) vs. 2.92 +/- 0.24% (P less than 0.05) and 0.97 +/- 0.14% vs. 1.72 +/- 0.15% (P less than 0.01), respectively]. The percentage of insulin in the intracellular compartment was also slightly lower in adipocytes from diabetic compared to normal subjects (0.84 +/- 0.19% vs. 1.20 +/- 0.16%; P greater than 0.05). The lysosomotropic agent chloroquine increased total cell-associated insulin, and this was due entirely to an increase in intracellular insulin. In adipocytes from normal subjects, chloroquine increased intracellular insulin by 32% at 30 min, by 89% at 60 min, by 140% at 90 min, and by 178% at 120 min. In comparison to the normal adipocytes, the chloroquine-mediated increase in intracellular insulin was lower in adipocytes from the diabetic subjects (-8.1% at 30 min, 37% at 60 min, 58% at 90 min, and 63% at 120 min; P less than 0.05 at all time points). These results indicate that insulin is rapidly internalized in human adipocytes at 37 C such that approximately half of total cell-associated insulin is intracellular the intracellular insulin is largely intact; and intracellular processing of insulin by a chloroquine-sensitive pathway(s) is impaired in adipocytes from type II diabetic subjects.