Receptor-linked degradation of 125I-insulin is mediated by internalization in isolated rat hepatocytes

When hepatocytes were freshly isolated from rat liver and incubated for various periods of time at 37 degrees C, the media from the incubation, when completely separated from the cells, actively degraded 125I-insulin. THis soluble protease activity was strongly inhibited by bacitracin but was unaffected by the lysosomatropic agent ammonium chloride (NH4Cl). When hepatocytes were incubated with 125I-insulin at 37 degrees C in the presence or absence of 8 mM NH4Cl the ligand initially bound to the plasma membrane and was subsequently internalized as a function of time. When hepatocytes were incubated at 37 degrees C for 30 minutes with 125I-insulin in the presence of bacitracin and NH4Cl or bacitracin alone and the cells were washed, diluted, and the cell-bound radioactivity allowed to dissociate, the percent intact 125I-insulin in the cell pellet and in the incubation media was greater in the presence of NH4Cl at each time point of incubation. Under these same conditions a higher proportion of the cell-associated radioactivity was internalized and a higher proportion was associated with lysosomes. The data suggest that receptor-mediated internalization is required for insulin degradation by the cell, and that this process, at least in part, involves lysosomal enzymes. Furthermore, the data demonstrate that internalization is not blocked by the presence of bacitracin or NH4Cl in the incubation media, but that degradation is inhibited.     

Binding and degradation of 125I-insulin by rat hepatocytes.

The binding and the velocity of degradation of 125I-insulin in the absence or presence of varying concentrations of native procline insulin were studied using isolated rat hepatocytes. At insulin concentrations ranging from 5 X 10(-11) to 10(-6) M, insulin degradation velocity showed a first order dependence on the total concentration of insulin bound at steady state. The overall reaction had an apparent rate constant of 0.030 +/- 0.011 min-1. Furthermore, the degradation of a given amount of 125I-insulin bound to cells was more rapid and extensive than the degradation of the same amount of insulin which had been newly exposed to fresh cells. Mid pretreatment of isolated hepatocytes with trypsin or chymotrypsin at concentrations of 5 to 20 mug/ml depressed to the same degree the amount of 125-I-insulin bound at steady state and the 125I-insulin degradation velocity. Peptide or protein hormones unrelated to insulin, including the oxidized A and B chains of insulin, failed to depress the amount of insulin bound or the velocity of insulin degradation when present at concentrations of 10-5 or 10-6 M. Over a wide range of concentrations, various synthetic insulin analogues and naturally occurring insulins depressed to the same degree the amount of 125I-insulin bound at steady state and the 125I-insulin degradation velocity. These observations suggest that insulin bound to hepatocyte plasma membranes is the substrate for insulin degradation by the liver.     

Peculiarities of distribution of 125I-insulin and 125I-insulin-like growth factor-I (IGF-I) at internalization in isolated rat hepatocytes

One of determining conditions of formation in vertebrate phylogenesis of hormonal systems of insulin and IGF-I—peptides common by origin, similar by structure and by biological action is temperature factor. In differentiation of functional roles of two related hormones an important place is ascribed to mechanism of their intracellular action. Study of formation of the two hormonal systems in vertebrate phylogenesis necessitates knowledge of peculiarities of internalization of two related hormones in mammals. On isolated rat hepatocytes at the identical maximal level of internalization of ^I25I-insulin and ¹²⁵I-IGF-I there are revealed marked differences of dynamics of their internalization. Besides, at internalization of ¹²⁵I-insulin or of ¹²⁵I-IGF-I, their peculiar distribution was observed in the cell and on the plasma membrane. At 37°C, only two thirds of ¹²⁵I-insulin relatively bound to receptors on the membrane were immersed into cell, whereas the portion of internalized ¹²⁵I-IGF-I turned out to be higher than the part located on the membrane. At 12°C, a decrease of ¹²⁵I-insulin inside the cell and its increase on the membrane indicated the interdependent label redistribution under these conditions. The form of the ¹²⁵I-IGF-I distribution at low temperature remained unchained. The pattern of established differences of internalization of ¹²⁵I-insulin and ¹²⁵I-IGF-I as well as different sensitivity of each of the processes to low temperature indicated that each of the peptides triggered individual mechanism of endocytosis of receptors. The high sensitivity of internalization of ¹²⁵I-insulin to low temperature and the temperature lability of the process in isolated rat hepatocytes agree with the earlier suggestion about the late formation of the regulatory insulin system in homoiothermal vertebrates.     

Specific 125I-insulin binding by hepatocyte plasma membranes and nuclear envelopes in experimental hyperinsulinemia, alloxan diabetes and starvation

A comparative study has been carried out of the insulin-receptor interaction in rat liver plasma membrane and nuclear envelope under experimental hyperinsulinemia (14 days) and hypoinsulinemic states (3 days alloxan diabetes, 2 days fasting, and joint action of these factors). In all the cases, including additivity of the receptorotropic effects of diabetes and fasting, the changes in levels of specific binding of 125I-insulin and indices, reflecting receptor number, were unidirectional both in the plasma membrane and nuclear envelope. Some divergences were observed for indices reflecting the affinity changes of the receptors from different sources. It is suggested that in insulin-induced regulation of insulin receptor number the relationship between intracellular membrane receptors and receptors in the plasma membrane and nuclear envelope is the same.     

Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study

Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.     

Uptake and degradation of 125I-labelled asialo-fetuin by isolated rat hepatocytes

¹²⁵I-Labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 · 10^?8M asialo-fetuin. Non-parenchymal liver cells did not take up asialo-fetuin in vitro. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Asialo-fetuin consequently accumulated in the cells until the extracellular supply was exhausted. Asialo-fetuin degradation could be studied without concurrent uptake by incubating cells, previously exposed to asialo-fetuin, in asialo-fetuin-free medium. Degradation, as evidenced by increase in acid-soluble radioactivity, was inhibited by NH₄Cl and chloroquine. The change with time in the intracellular distribution pattern of radioactivity in cells that had been exposed to ¹²⁵I-labelled asialo-fetuin for 10 min was examined by means of differential centrifugation. Initially, the radioactivity was found mostly in the microsomal fraction. 60 min after the exposure to labelled protein, the distribution pattern of radioactivity resembled that of the lysosomal enzyme β-acetylglucosaminidase. The possibility that asialo-fetuin digestion takes place in lysosomes is discussed.     

Studies on the effects of alloxan and streptozotocin induced diabetes on lipid metabolism in the isolated perfused rat lung.

The effects of experimentally induced diabetes on the conversion of glucose to lipid in the isolated perfused rat lung were examined. Alloxan diabetes and streptozotocin diabetes reduced the incorporation of glucose into the neutral lipid and phospholipid fractions of the lung to a rate less than 40% of that observed in normal animals. This phenomenon appears to be related to insulin deficiency as lungs from diabetic rats treated for one week with insulin were capable of incorporating glucose at a rate comparable to that observed in normal animals. While insulin $\text{in}$$\text{vivo}$ altered lipid metabolism in perfused lung, $\text{in}$$\text{vitro}$ insulin had no demonstrable effect on lipid metabolism in the perfused lung, an indication that the effects of the hormone may be long term rather than short term. These data indicate that pulmonary lipid metabolism may be regulated by the action of insulin.     

Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

Uptake and degradation of 125I-labelled asialo-fetuin by isolated rat hepatocytes.

125I-Labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 - 10(-8) M asialo-fetuin. Non-parenchymal liver cells did not take up asialo-fetuin in vitro. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Asialo-fetuin consequently accumulated in the cells until the extracellular supply was exhausted. Asialo-fetuin degradation could be studied without concurrent uptake by incubating cells, previously exposed to asialo-fetuin, in asialo-fetuin-free medium. Degradation, as evidenced by increase in acid-soluble radioactivity, was inhibited by NH4Cl and chloroquine. The change with time in the intracellular distribution pattern of radioactivity in cells that had been exposed to 125I-labelled asialo-fetuin for 10 min was examined by means of differential centrifugation. Initially, the radioactivity was found mostly in the microsomal fraction. 60 min after the exposure to labelled protein, the distribution pattern of radioactivity resembled that of the lysosomal enzyme beta-acetylglucosaminidase. The possibility that asialo-fetuin digestion takes place in lysosomes is discussed.     

Effect of cell density on metabolism in isolated rat hepatocytes

Freshly isolated rat hepatocytes show many changes in metabolic activities as a function of cell density in the incubation flask. Fatty acid synthesis, cholesterol synthesis, general protein synthesis, and rates of accumulation of pyruvate, lactate, citrate, acetyl-CoA, acetoacetate and beta-hydroxybutyrate decrease as the cell density increases between 1 mg/ml and 60 mg/ml. Glucose release only decreases between 1-5 mg/ml and the concentration of ATP does not vary at any density. There is a small increase in the lactate/pyruvate ratio and a dramatic decrease in the beta-hydroxybutyrate/acetoacetate ratio with increasing cell concentration. When cells at 8 or 28 mg/ml were incubated with added lactate and pyruvate, or alanine, a two fold increase in fatty acid synthesis and 50% decrease in cholesterol synthesis were observed as compared to rates with endogenous substrate. With added glucose the synthetic rates were similar to those obtained with endogenous substrate. However, regardless of the type of substrate used, the less dense cells gave rates up to three times greater than that of the more dense cells. When conditioned medium isolated after incubation of cells at high density was added to the less dense cells, a decrease in the rates of fatty acid synthesis and cholesterol synthesis was observed in the less dense cells; however, when medium from the less dense cells after incubation for the same period was added to the more dense cells, there was no significant change in fatty acid or cholesterol synthesis. These results suggest that a factor may be released into the medium of incubating hepatocytes that progressively inhibits certain metabolic processes as the cell density increases.     

Studies on gluconeogenesis and ketogenesis in isolated hepatocytes from alloxan diabetic rats.

Gluconeogenesis and ketogenesis were studied in isolated hepatocytes obtained from normal and alloxan diabetic rats. Insulin treatment maintained near-normal blood glucose levels and caused an increase in glycogen deposition. The third day after insulin withdrawal the rats displayed a diabetic syndrome marked by progressive hyperglycemia and glycogen depletion. Net glucose production in liver cells isolated from alloxan diabetic rats progressively increased with time up to 72 hr after the last in vivo insulin injection. Maximal glucose production was observed at 72 hr with 10 mM alanine, lactate, pyruvate, or fructose. Glucose production decreased at 96 hr. The same pattern was observed with the incorporation of labeled bicarbonate into glucose. Ketogenesis in liver cells and hepatic lipid content also peaked at 72 hr.