Receptor- and non-receptor-mediated uptake and degradation of insulin by hepatocytes

Native insulin inhibits the binding and degradation of ¹²⁵I-labelled insulin in parallel. Half-maximal inhibition of degradation occurs with 10nm-insulin, a hormone concentration sufficient to saturate the insulin receptor. The proportion of bound hormone that is degraded increases as the insulin concentration is increased, suggesting that low-affinity uptake is functionally related to degradation. Since only a small fraction (approx. 10%) of the overall degradation occurs at the plasma membrane, or in the extracellular medium, translocation of bound hormone into the cell is the predominant mechanism mediating the degradation of insulin. In the presence of 0.6nm-insulin, a concentration at which most cell-associated hormone is receptor-bound, chloroquine increases the amount of ¹²⁵I-labelled insulin retained by hepatocytes. However, chloroquine increases the retention of degradation products of insulin in incubations containing sufficient hormone (6nm) to saturate the receptor and permit occupancy of low-affinity sites. Glucagon does not compete for the interaction of ¹²⁵I-labelled insulin (1nm) with the insulin receptor. In contrast, 20μm-glucagon inhibits 75% of the uptake of insulin (0.1μm) by low-affinity sites. A fraction of the cell-bound radioactivity is not intact insulin throughout a 90min association reaction at 37°C. During dissociation, fragments of ¹²⁵I-labelled insulin are released to the medium more rapidly than is intact hormone. The production and transient retention of degradation products of the hormone complicates the characterization of the insulin receptor by equilibrium or kinetic methods of assay. It is proposed that insulin degradation occurs by receptor- and non-receptor-mediated pathways. The latter may be related to the action of glutathione–insulin transhydrogenase, with which both insulin and glucagon interact.     

Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells.

The effects of various agents on the binding and degradation of 125I-labelled insulin by isolated rat hepatocytes and cultured H4 hepatoma cells were studied. Various lysosomotropic agents, including chloroquine, ammonium chloride, and the topical anesthetics, lidocaine and procaine inhibited insulin degradation by H4 hepatoma cells but had little effect on the binding of the hormone. Similarly, tosyl-L-lysyl chloromethyl ketone selectively inhibited the degradation of 125I-labelled insulin by isolated hepatocytes, as did the sulfhydryl reagents, p-hydroxy- and p-chloromercuriphenyl sulfonic acid. Inhibitors of energy production, including sodium fluoride, sodium azide, and dinitrophenol, also selectively inhibited the degradation of insulin by hepatocytes, although cyanide had no effect under the conditions used. Lectins and antimicrotubular agents, which are known to affect the mobility of plasma membrane proteins or of intracytoplasmic vesicles, selectively inhibited insulin degradation by hepatocytes to varying degrees, whereas agents which inhibit the function of microfilaments had no effect. At temperatures below 20 degrees C, insulin degradation was negligible but rose rapidly between 20 and 37 degrees C, suggesting that a membrane-related step is rate limiting in the overall degradative process. These results are all consistent with a model of insulin uptake by target tissue involving pinocytosis of receptor-bound hormone followed by intralysosomal degradation.     

Proteolytic degradation of insulin and glucagon.

The degradation of insulin and glucagon by a highly purified enzyme isolated from rat skeletal muscle was investigated. A sensitive assay for proteolytic degradation of insulin and glucagon using fluorescamine to detect an increase in primary amine groups was established. As measured by an increase in fluorescamine reactive materials, insulin was rapidly degraded by this highly purified enzyme without requiring initial disulfide cleavage. Associated with the increase in fluorescamine reactive materials was a decrease in immunoassayable insulinmglucagon wal also proteolytically degraded by this enzyme but a number of other peptides and proteins including proinsulin, and A and B chains of insulin were not degraded. Thus, we have demonstrated that insulin (and glucagon) can be proteolytically degraded by an enzyme isolated from an insulin sensitive tissue, skeletal muscle. Proteolytic degradation by this enzyme requires the intact insulin molecule rather than separate A and B chains.     

Direct uptake and degradation of DNA by lysosomes

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered “RNautophagy,” an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term “DNautophagy,” is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa.     

Natural regulatory mechanisms of insulin degradation by insulin degrading enzyme

Insulin-degrading enzyme (IDE) accounts for most of the insulin degrading activity in extracts of several tissues and plays an important role in the intracellular degradation of insulin. Using newly developed sandwich radioimmunoassay for rat IDE, this enzyme was detectable in all tissues we examined and liver had the highest level of IDE. The ratio of insulin degrading activity to IDE concentration was roughly the same in liver, brain and muscle, however, twice as high in kidney as compared with other tissues. On the contrary, its degrading activity in these tissue extracts, including kidney, was completely lost after immunoprecipitation of IDE. These results suggest that IDE degrades insulin in the initial step of cleavage and that there are some mechanisms to regulate insulin degrading activity by IDE in the tissues.     

CD44-mediated uptake and degradation of hyaluronan.

Hyaluronan turnover occurs systemically from the lymph and serum as well as locally by the same cells responsible for its synthesis. Local turnover involves receptor-mediated uptake and delivery to lysosomes. Of the many hyaluronan binding proteins/receptors known, the participation of CD44 in the internalization of hyaluronan has been best characterized. Some fraction of the hyaluronan bound to CD44 becomes internalized and delivered to lysosomes by a mechanism that is not dependent on clatherin, caveolae or pinocytosis. In cells such as chondrocytes, anabolic and catabolic cytokines can alter the activity of CD44 toward hyaluronan internalization. However, the mechanism of cellular regulation remains unclear. Regulation may involve the participation of alternatively spliced isoforms of CD44, changes in CD44 phosphorylation, changes in cytoskeletal binding proteins or, the activity or extracellular proteolytic activity.     

Ras inhibition induces insulin sensitivity and glucose uptake

Background

Reduced glucose uptake due to insulin resistance is a pivotal mechanism in the pathogenesis of type 2 diabetes. It is also associated with increased inflammation. Ras inhibition downregulates inflammation in various experimental models. The aim of this study was to examine the effect of Ras inhibition on insulin sensitivity and glucose uptake, as well as its influence on type 2 diabetes development.

Methods and Findings

The effect of Ras inhibition on glucose uptake was examined both in vitro and in vivo. Ras was inhibited in cells transfected with a dominant-negative form of Ras or by 5-fluoro-farnesylthiosalicylic acid (F-FTS), a small-molecule Ras inhibitor. The involvement of IκB and NF-κB in Ras-inhibited glucose uptake was investigated by immunoblotting. High fat (HF)-induced diabetic mice were treated with F-FTS to test the effect of Ras inhibition on induction of hyperglycemia. Each of the Ras-inhibitory modes resulted in increased glucose uptake, whether in insulin-resistant C2C12 myotubes in vitro or in HF-induced diabetic mice in vivo. Ras inhibition also caused increased IκB expression accompanied by decreased expression of NF-κB . In fat-induced diabetic mice treated daily with F-FTS, both the incidence of hyperglycemia and the levels of serum insulin were significantly decreased.

Conclusions

Inhibition of Ras apparently induces a state of heightened insulin sensitization both in vitro and in vivo. Ras inhibition should therefore be considered as an approach worth testing for the treatment of type 2 diabetes.     

Receptor-mediated degradation of insulin in isolated rat adipocytes: Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [Tyr^A14-¹²⁵I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [¹²⁵I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the ‘insulin’ peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

Receptor-mediated degradation of insulin in isolated rat adipocytes. Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [TyrA14-125I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [125I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the 'insulin' peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

Effects of insulin on cardiac lysosomes and protein degradation

Hearts perfused in the absence of added insulin had 1) accelerated rates of protein degradation, as assessed by release of phenylalanine and tyrosine; 2) increased rates of release of seven other amino acids; 3) decreased lysosomal latency and sedimentable lysosomal enzyme activity; 4) increased numbers of autophagic vacuoles in cardiac muscle cells; and 5) decreased activity of beta-N-acetylglucosaminidase in dense lysosomes (1.06-1.09 g/ml), as compared to hearts perfused in the presence of the hormone. After 3 h of perfusion in the absence of insulin, the changes that developed in protein degradation, lysosomal latency, and sedimentability, and in enzyme activity in dense lysosomes, were reversed by insulin addition during 90 min of subsequent perfusion. These studies suggest a role for insulin in controlling the activity of the lysosomal system and the involvement of this system in protein degradation, particularly in insulin-deprived tissue.     

Effect of internalization and degradation of insulin on rat adipocyte insulin receptor binding kinetics

We have assessed the influence of nondisplaceable (internalized) insulin and insulin degradation during binding reactions at 37 degrees C on the numbers and affinities of insulin binding sites on isolated rat adipocytes. Corrections for nondisplaceable insulin caused a 33% reduction in the number of the high affinity sites (p less than 0.01) and a 24% reduction (p less than 0.01) in the number of the low affinity sites which was associated with a 20% increase (p less than 0.01) in affinity when a two-site model was applied. With a one-site model, the number of insulin receptors decreased by approximately 33% (p less than 0.01), but the affinity did not change. These results indicate that the internalization and degradation of insulin that occurs during the binding reaction can significantly affect the estimation of insulin binding kinetics. Potential variations in internalization and degradation of insulin by cells obtained under various physiological or pathologic conditions should, therefore, be taken into consideration in the interpretation of insulin binding data.     

Participation of cellular thiol/disulphide groups in the uptake, degradation and bioactivity of insulin in primary cultures of rat hepatocytes.

The effects on the uptake (cell-associated 125I) and degradation (125I-labelled products released into the medium) of 125I-insulin and bioactivity (protein, glycogen and lipid synthesis) of insulin caused by altering the cellular thiol/disulphide status in primary cultures of rat hepatocytes were studied. Incubation of hepatocyte cultures with various exogenous thiol compounds (reduced glutathione, 2-mercaptoethanol, cysteamine, dithiothreitol) resulted in increased insulin binding, but markedly decreased degradation and bioactivity. These effects could be reversed by washing or by the addition of oxidized glutathione, which alone had no effect. When cultures were exposed to certain thiol-modifying reagents (N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzenesulphonate, iodoacetamide, iodoacetate), some decreases in bioactivity were evident, but the pronounced decrease in insulin degradation observed with the thiol-containing compounds was not observed with this class of compounds. None of the thiol-containing or -modifying agents tested had any significant effect on cellular ATP concentrations, indicating that the effects observed were due to perturbation of the thiol/disulphide status. Depletion of intracellular glutathione by DL-buthionine SR-sulphoximine (a specific inhibitor of glutathionine biosynthesis) decreased the syntheses of glycogen and lipid by about one-half, while having essentially no effect on protein synthesis, ATP concentrations or on the binding and degradation of insulin. The data presented here indicate that although intracellular thiol (glutathione) concentrations may be important for the maintenance of full expression of certain biological activities (glycogen and lipid synthesis), the thiol/disulphide groups on the cell surface and those immediately inside the cell membrane may be more critical in the mediation of insulin action, including the degradation and bioactivity of insulin in primary cultures of rat hepatocytes.     

Swainsonine inhibits macrophage receptor-mediated uptake and degradation of a mannosyl-oligosaccharide

Rat pulmonary macrophages were incubated in the presence of a radiolabeled mannosyl-oligosaccharide obtained from ovalbumin. Receptor-mediated endocytosis and degradation of this ligand by the cells was followed in the presence or absence of swainsonine, an inhibitor of alpha-mannosidases. The results indicated that at higher concentrations (greater than 1 microgram/ml) of swainsonine, both the internalization and degradation of the radiolabeled ligand were inhibited. At a concentration of 0.1 microgram/ml of swainsonine, only the degradation was inhibited while the uptake was unaltered. The degradation of the oligosaccharide was blocked due to the inhibition of lysosomal alpha-mannosidase. However, the inhibition of lysosomal alpha-mannosidase was reversible upon withdrawal of swainsonine.     

Glucose-induced insulin resistance of skeletal-muscle glucose transport and uptake.

The ability of glucose and insulin to modify insulin-stimulated glucose transport and uptake was investigated in perfused skeletal muscle. Here we report that perfusion of isolated rat hindlimbs for 5 h with 12 mM-glucose and 20,000 microunits of insulin/ml leads to marked, rapidly developing, impairment of insulin action on muscle glucose transport and uptake. Thus maximal insulin-stimulated glucose uptake at 12 mM-glucose decreased from 34.8 +/- 1.9 to 11.5 +/- 1.1 mumol/h per g (mean +/- S.E.M., n = 10) during 5 h perfusion. This decrease in glucose uptake was accompanied by a similar change in muscle glucose transport as measured by uptake of 3-O-[14C]-methylglucose. Simultaneously, muscle glycogen stores increased to 2-3.5 times initial values, depending on fibre type. Perfusion for 5 h in the presence of glucose but in the absence of insulin decreased subsequent insulin action on glucose uptake by 80% of the effect of glucose with insulin, but without an increase in muscle glycogen concentration. Perfusion for 5 h with insulin but without glucose, and with subsequent addition of glucose back to the perfusate, revealed glucose uptake and transport similar to initial values obtained in the presence of glucose and insulin. The data indicate that exposure to a moderately increased glucose concentration (12 mM) leads to rapidly developing resistance of skeletal-muscle glucose transport and uptake to maximal insulin stimulation. The effect of glucose is enhanced by simultaneous insulin exposure, whereas exposure for 5 h to insulin itself does not cause measurable resistance to maximal insulin stimulation.     

Depot-specific regulation of glucose uptake and insulin sensitivity in HIV-lipodystrophy

Altered fat distribution is associated with insulin resistance in HIV, but little is known about regional glucose metabolism in fat and muscle depots in this patient population. The aim of the present study was to quantify regional fat, muscle, and whole body glucose disposal in HIV-infected men with lipoatrophy. Whole body glucose disposal was determined by hyperinsulinemic clamp technique (80 mU x m(-2) x min(-1)) in 6 HIV-infected men and 5 age/weight-matched healthy volunteers. Regional glucose uptake in muscle and subcutaneous (SAT) and visceral adipose tissue (VAT) was quantified in fasting and insulin-stimulated states using 2-deoxy-[18F]fluoro-D-glucose positron emission tomography. HIV-infected subjects with lipoatrophy had significantly increased glucose uptake into SAT (3.8 +/- 0.4 vs. 2.3 +/- 0.5 micromol x kg tissue(-1) x min(-1), P < 0.05) in the fasted state. Glucose uptake into VAT did not differ between groups. VAT area was inversely related with whole body glucose disposal, insulin sensitivity, and muscle glucose uptake during insulin stimulation. VAT area was highly predictive of whole body glucose disposal (r2 = 0.94, P < 0.0001). This may be mediated by adiponectin, which was significantly associated with VAT area (r = -0.75, P = 0.008), and whole body glucose disposal (r = 0.80, P = 0.003). This is the first study to directly demonstrate increased glucose uptake in subcutaneous fat of lipoatrophic patients, which may partially compensate for loss of SAT. Furthermore, we demonstrate a clear relationship between VAT and glucose metabolism in multiple fat and muscle depots, suggesting the critical importance of this depot in the regulation of glucose and highlighting the significant potential role of adiponectin in this process.     

Characterization of insulin degradation by rat-liver low-density vesicles

When incubated in vitro, isolated rat liver low-density vesicles degrade endocytosed insulin intraluminally. The rate of intravesicular degradation suggests that this pathway contributes significantly to insulin degradation in vivo. The vesicles can be selectively disrupted with digitonin at concentrations that abolish the latency of NADH pyrophosphatase, with minimal effect on the cisternal Golgi marker, galactosyl transferase. The results suggest that latent NADH pyrophosphatase may act as a marker enzyme for the vesicles within which insulin is degraded. The possible role of insulin-glucagon protease, a candidate enzyme for insulin degradation by the liver, was investigated. The activity of latent insulin-glucagon protease associated with low-density vesicles is sufficient to account for the rate of intravesicular proteolysis. However, the rate of intravesicular proteolysis is insensitive to membrane-permeant thiol reagents under conditions which strongly inhibit insulin-glucagon protease. This shows that insulin-glucagon protease is not rate-limiting for insulin degradation by these vesicles, and is unlikely to be involved in the regulation of degradation. After disruption with Brij, internalized insulin remains associated with the membrane. Degradation is not inhibited by addition of excess unlabelled insulin to the medium, and occurs more rapidly than the degradation of an equal activity of iodo-insulin added to the disrupted membranes. This implies that degradation of endocytosed insulin occurs while it is still bound to the inner surface of the vesicles. When bacitracin is coinjected with iodo-insulin, it inhibits degradation of internalized insulin both by intact and Brij-disrupted vesicles, but not the degradation of added exogenous insulin, confirming that degradation is membrane-associated, and that it does not require the release of insulin into free solution.