Insulin Internalization and Degradation in Adipocytes from Normal and Type II Diabetic Subjects*

Abstract

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.

The Sulfonylurea Glimepiride Regulates Intracellular Routing of the Insulin-Receptor Complexes through Their Interaction with Specific Protein Kinase C Isoforms

“…Previous in vivo and in vitro investigations have provided substantial evidence for a relationship between insulin action and intracellular processing of insulin and its receptor (Ferrannini et al, 1982;Flier et al, 1982;Peavy et al, 1984;Veda et al, 1985;Jochen and Berhanu, 1987;Miller, 1988). Furthermore, it has been suggested that abnormalities in insulin receptor recycling and intracellular processing of the insulinreceptor complex might contribute to impair insulin action in patients with type 2 diabetes mellitus (Jochen et al, 1986;Grunberger et al, 1989;Trischitta et al, 1989;Benzi et al, 1990Benzi et al, , 1997Sesti et al, 1996). We found that treatment with glimepiride causes an increase in both insulin sensitivity and responsiveness for glucose incorporation into glycogen.…”

“…Receptor-mediated insulin endocytosis is the principal mechanism for insulin clearance from the blood, and evi- dence has been provided that this process is impaired in patients with type 2 diabetes (Jochen et al, 1986;Grunberger et al, 1989;Trischitta et al, 1989;Benzi et al, 1990Benzi et al, , 1997. Thus, regulation by sulfonylureas of intracellular processing of insulin might have profound pathophysiological consequences by modulating the concentration of insulin in the peripheral circulation and avoiding the deleterious effects of hyperinsulinemia.…”

“…Association between impaired in vivo insulin clearance and action have been reported in subjects with insulin resistance (Ferrannini et al, 1982;Flier et al, 1982). Moreover, defects in insulin-receptor internalization and processing have been reported in various cells from patients with type 2 diabetes mellitus including circulating monocytes (Grunberger et al, 1989;Trischitta et al, 1989;Benzi et al, 1990;Benzi et al, 1997), isolated adipocytes (Jochen et al, 1986) and cultured Epstein Barr virus-transformed lymphocytes (Sesti et al, 1996), thus suggesting that abnormalities of these processes might contribute to the insulin resistance of type 2 diabetes. Overall, these findings raise the possibility that a sulfonylurea-induced improvement in intracellular insulin degradation may explain the decrease in plasma insulin levels associated with improved glucose tolerance observed in patients with type 2 diabetes chronically treated with sulfonylureas (Kolterman et al, 1984).…”

Adipocyte Induction of Preadipocyte Differentiation in a Gradient Chamber

“…14 Jochen and coworkers reported that the amount of insulin internalized by a suspension of freshly isolated human adipocytes peaked after 30 min of incubation at 37°C. 15 By this time, 40% of the total insulin associated with the cells was internalized. After 60 min, the percentage of total cellassociated insulin within the cells dropped to 1.2%.…”

Reversibility of defective adipocyte insulin receptor kinase activity in non-insulin-dependent diabetes mellitus. Effect of weight loss.

“…To date, a host ofcellular abnormalities have been described in NIDDM, including decreased binding of insulin to its receptor (1, 2, 5, 7-1 1), diminished rate of insulin receptor internalization (12), defects in the glucose transport system (1, 5, 9-11, 13, 14), and defects in the action ofintracellular enzymes (15). Whereas these alterations are part of the disordered carbohydrate and insulin metabo-lism observed in NIDDM, it is not clear that any of these defects represent a primary alteration.…”