Human red blood cell insulin-degrading enzyme and rat skeletal muscle insulin protease share antigenic sites and generate identical products from insulin.

The mechanisms of cellular insulin degradation remain uncertain. Considerable evidence now exists that the primary cellular insulin-degrading activity is a metallothiol proteinase. Two similar degrading activities have been purified and characterized. Insulin protease has been purified from rat skeletal muscle and insulin-degrading enzyme from human red blood cells. Whereas the two degrading activities share a number of similar properties, significant differences have also been reported; and it is not at all established that they are the same enzyme. To examine this, we have compared antigenic and catalytic properties of the two enzymatic activities. Monoclonal antibodies against the red blood cell enzyme adsorb the skeletal muscle enzyme; and on Western blots, the antibodies react with an identical 110-kDa protein. Immunoaffinity-purified enzymes from both red blood cells and skeletal muscle degrade [125I]iodo(B26)insulin to the same products as seen with purified insulin protease and with intact liver and kidney. Chelator-treated muscle and red blood cell enzymes can be reactivated with either Mn2+ or Ca2+. Thus, insulin-degrading enzyme and insulin protease have similar properties. These results support the hypothesis that these activities reside in the same enzyme.     

Purification and autophosphorylation of insulin receptors from rat skeletal muscle

Insulin receptors of rat skeletal muscle were purified by first extracting a plasma membrane-enriched pellet obtained from a muscle homogenate with Triton X-100, followed by WGA-Sepharose and insulin-Sepharose affinity chromatography. Routinely, 4-5 micrograms of purified receptor were obtained from 15 g of tissue. The purified receptors are composed of two major polypeptides with molecular weights of 130,000 and 95,000, respectively. The binding of [125I]insulin by the purified receptors was analyzed by a Scatchard plot. There are at least two binding components. The high-affinity component, with an apparent association constant (Ka) of 2.0 X 10(9) M-1, comprises 10% of the total insulin binding sites; while the low-affinity component, with a Ka value of 1.4 X 10(8) M-1, represents 90% of the binding sites. Assuming the insulin receptor to have a molecular weight of 300,000, the receptor binds 1.7 mol of insulin per mol at saturation. Insulin is capable of stimulating the autophosphorylation of the beta-subunit of the muscle insulin receptor (Mr 95,000) by 5-10-fold. The stoichiometry of this phosphorylation reaction was determined as 0.8 phosphate per insulin binding site after a 10 min incubation with 100 nM insulin. In a previous report, I showed that the insulin stimulation of glucose transport in diaphragms from neonatal rats was small, even although the diaphragms had normal levels of insulin receptors and glucose transporters (Wang, C. (1985). Proc. Natl. Acad. Sci. USA 82, 3621-3625). To determine whether or not receptor autophosphorylation might be related to this insensitivity to insulin, the level of receptor phosphorylation was quantitated in diaphragms from rats at different stages of development. Autophosphorylation remains unchanged from birth to 21 days of age, suggesting that the lower insulin-stimulated glucose uptake by diaphragms at early stages of postnatal development as compared to that by diaphragms of older rats, is not due to a difference in receptor kinase.     

Rapid hyperpolarization of rat skeletal muscle induced by insulin

It has been proposed that the increase produced by insulin in electrical potential differences across membranes of target cells may be a mechanism by which the cell surface insulin-receptor complex causes at least some of the metabolic effects of insulin. If insulin-induced hyperpolarization is a transducer of common effector responses it must precede those responses. The problem has not been addressed previously, so that rapid responses to insulin have not been sought. Two methods were used. In one method, the bathing solution was changed rapidly so as to include insulin in supramaximal concentrations, and a series of measurements of membrane potentials, E_r, were made. Insulin hyperpolarized by 9.4 mV within 1 min. In the other method, nanoliter amounts of highly concentrated insulin solution were ejected from a micropipette onto the surface of an impaled muscle fiber. In 21 out of 32 insulin injections, hyperpolarization occurred within 1 s; in 11 control injections there was no change. This is the most rapid response to insulin yet reported, and is consistent with the hypothesis that insulin-induced hyperpolarization may transduce effector responses.     

Rat brain insulin degrading enzyme in insulin and thyroid hormones imbalances

Rat brain insulin degrading enzyme activity and its relationship with insulin receptor were investigated in experimental hyperglycemia, hyperinsulinemia, hypothyroidism and hyperthyroidism. Insulin degrading enzyme activity was assessed in synaptosomes and high speed cytosol using [125I]insulin. Levels of insulin degrading enzyme were changed in high speed cytosol in insulin and thyroid hormone imbalances. These results suggest that insulin degrading enzyme in brain is predominantly active in cytosol and is subject to regulation by insulin and thyroid hormones. Probably it plays some role in long term effects of insulin in brain.     

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.     

Stem cell antigen-1 localizes to lipid microdomains and associates with insulin degrading enzyme in skeletal myoblasts

Stem cell antigen-1 (Sca-1, Ly6A/E) is a glycosylphosphotidylinositol-anchored protein that identifies many tissue progenitor cells. We originally identified Sca-1 as a marker of myogenic precursor cells and subsequently demonstrated that Sca-1 regulates proliferation of activated myoblasts, suggesting an important role for Sca-1 in skeletal muscle homeostasis. Beyond its functional role in regulating proliferation, however, little is known about the mechanism(s) that drive Sca-1-mediated events. We now report that lipid microdomain organization is essential for normal myogenic differentiation, and that Sca-1 constitutively localizes to these domains during myoblast proliferation and differentiation. We also demonstrate that Sca-1 associates with insulin degrading enzyme (IDE), a catalytic protein responsible for the cleavage of mitogenic peptides, in differentiating myoblasts. We show that chemical inhibition of IDE as well as RNAi knockdown of IDE mRNA recapitulates the phenotype of Sca-1 interference, that is, sustained myoblast proliferation and delayed myogenic differentiation. These findings identify the first signaling protein that physically and functionally associates with Sca-1 in myogenic precursor cells, and suggest a potential pathway for Sca-1-mediated signaling. Future efforts to manipulate this pathway may lead to new strategies for augmenting the myogenic proliferative response, and ultimately muscle repair.     

Characterization of rat skeletal muscle sarcolemmal insulin receptors and a sarcolemmal insulin binding inhibitor

When insulin receptors of rat skeletal muscle sarcolemmal vesicles were solubilized with Triton X-100, the specific binding of 125I-labeled insulin increased by more than 10-fold over that seen in the intact vesicles. Partial purification of the skeletal muscle insulin receptors on wheat germ agglutinin affinity columns increased the total insulin binding activity by 7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM, suggesting that an inhibitor of insulin binding was removed by this purification step. This was confirmed when the unbound fractions of the affinity column were dialyzed and reconstituted with the insulin receptors. The inhibitory activity in the sarcolemmal extract could not be accounted for by the presence of Triton X-100. The skeletal muscle inhibitor was more potent in inhibiting insulin binding to skeletal muscle insulin receptors than to liver or adipose receptors. The inhibitor was very effective in inhibiting insulin binding to wheat germ agglutinin-purified IM-9 receptors, but had negligible effects on insulin binding to intact IM-9 cells. The properties of the alpha and beta subunits of the skeletal muscle insulin receptors appear to be the same as those of insulin receptors of other tissues: cross-linking of 125I-labeled insulin to the receptor revealed a band of 130,000 daltons, and insulin stimulated the phosphorylation of bands of 90,000 and 95,000 daltons in the receptor preparation. The skeletal muscle insulin binding inhibitor elutes from molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol, or by trypsin, pepsin, or proteinase K. Collectively, these data suggest that the inhibitor may be a small molecule that aggregates with itself, with larger proteins, or with detergent micelles.     

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.     

Mosaic lectin and enzyme staining patterns in rat skeletal muscle.

We compared the localizations of lectin binding and activity for myosin ATPase and succinic dehydrogenase in sections of the gracilis, soleus, and masseter muscles from 10- and 60-day-old rats. In the 60-day-old rats, incubation of the muscle sections with the lectins ConA, GS-II, HPA, and jacalin gave rise to a mosaic staining pattern, especially in the gracilis muscle, in which the same fibers were strongly stained for ConA, GS-II, and HPA, whereas the staining with jacalin in these fibers was weak, and vice versa. There was no correspondence in the staining patterns for the enzymes and the lectins. In the masseter muscle only GS-II gave rise to distinct differences in the staining intensity between muscle fibers. In 10-day-old rats all fibers in the muscles were moderately stained with ConA, HPA, and jacalin, whereas a chessboard staining pattern could be observed after incubation with GS-II. In an extract of hindleg muscle from 60-day-old rats there was strong affinity for ConA and HPA and weak affinity for GS-II and jacalin, as shown by dot-blotting. After electrophoresis and blotting to nitrocellulose membranes, three muscle protein bands with apparent molecular weights of 100,000, 90,000, and 43,000 showed affinity for ConA, HPA, and GS-II, whereas no bands were jacalin positive. The complex lectin staining pattern in skeletal muscle might be related to development, specialization, and function of the muscles.     

How the binding and degrading capabilities of insulin degrading enzyme are affected by ubiquitin

Insulin degrading enzyme (IDE) is known to play a pivotal role on amyloidogenic peptide degradation but little is known about the changes in the proteolytic activity of the enzyme upon modification of external factors. Particularly, although it has been reported that altered ubiquitin concentration and/or hyperinsulinaemia increase the risk of developing Alzheimer's disease (AD), the molecular mechanism involved is unclear. In this work, we study the role that ubiquitin plays on IDE capability of binding and degrading insulin molecules and the obtained results indicate that ubiquitin has an allosteric role for IDE and high ubiquitin levels impair IDE activity.     

Crystallization and amino acid composition of a serine protease from rat skeletal muscle

A serine protease from rat skeletal muscle was crystallized in good yield, and the homogeneity of the preparation was proved by ultracentrifugical analysis and polyacrylamide disc electrophoresis. The S_{20, w} value of the enzyme was 2.2 S and the molecular weight was calculated to be 22,000–24,000 from the results of sedimentation equilibrium analysis. Analysis showed 87% coincidence in the amino acid composition with that of a serine protease from the small intestine. The apparent Km and k_cat(sec^?1) values for N-acetyl-L-tyrosine ethyl ester were 1.1 × 10^?3 M and 9.0, respectively.     

Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney

We have identified a metalloendoprotease from rat kidney cortex that cleaves the cysteine-phenylalanine bond (Cys7-Phe8) within the 17 amino acid ring structure of atrial natriuretic factor (ANF). Cleavage at this site represents the major ANF degradative activity in rat kidney, and is inhibited by the known metalloendoprotease inhibitors, thiorphan, phosphoramidon and zincov with IC50 values in the nanomolar range. Since these are specific inhibitors of protease 3.4.24.11, both protease 3.4.24.11 and ANF degrading activities were monitored during purification. Both activities copurified at each chromatographic step. Furthermore, purified protease 3.4.24.11 cleaved ANF specifically at the Cys7-Phe8 bond. It is concluded from this work that the major ANF degrading enzyme in rat kidney is protease 3.4.24.11.     

Glucose transport into rat skeletal muscle: interaction between exercise and insulin

This study was done to evaluate the effect of insulin on sugar transport into skeletal muscle after exercise. The permeability of rat epitrochlearis muscle to 3-O-methylglucose (3-MG) was measured after exposure to a range of insulin concentrations 30, 60, and 180 min after a bout of exercise. Thirty and 60 min after exercise, the effects of exercise and insulin on 3-MG transport were additive over a wide range of insulin concentrations, with no increase in sensitivity or responsiveness to insulin. After 180 min, when approximately 66% of the exercise-induced increase in sugar transport had worn off, both the responsiveness and sensitivity of the glucose transport process to insulin were increased. These findings appear compatible with the hypothesis that the actions of exercise and insulin result in activation and/or translocation into the plasma membrane of two separate pools of glucose transporters in mammalian skeletal muscle.     

Carboxylation of pyruvate via extramitochondrial malic enzyme in rat skeletal muscle

Soluble rat skeletal muscle extract incubated in the presence of pyruvate, NaH14CO3, MgCl2 and NADP-generating system fixed H14CO3- at the rate of about 2.3 nmol/min per mg protein. The omission of pyruvate or NADPH-generating system abolished completely the fixation of H14CO3-. This suggests that malic enzyme is responsible for the carboxylation of pyruvate. A possible role of pyruvate carboxylation by extramitochondrial malic enzyme in the conversion of lactate into glycogen in skeletal muscle is discussed.     

Glycogen synthase in diabetic rat skeletal muscle: activation by insulin

Summary Glycogen synthase in skeletal muscle of 3-day alloxan-diabetic rats was found to be in a less active state than in normal muscle. Both the activity ratio (activity without G6P divided by activity with 7.2 mM G6P at 4.4 mM UDPG, pH 7.8) and fractional velocity (activity with 0.25 mM G6P divided by activity with 10 mM G6P at 0.03 mM UDPG, pH 6.9) were significantly lower in the diabetic tissue. Correspondingly, the S_0.5 for UDPG and A_0.5 for G6P were significantly higher in diabetic tissue, suggesting decreased affinity for substrate and activator, respectively. The kinetic changes in the diabetic synthase were identical whether the alloxan-treated animals were maintained on insulin for 7 days prior to withdrawal for 3 days, or studied 3 days immediately after alloxan treatment. The diabetes-induced changes in synthase could be reversed by injecting the diabetic rat with insulin 10 min prior to sacrifice. After insulin treatment, the S_0.5 for UDPG and A_0.5 for G6P decreased to control levels or lower and the activity ratios and fractional velocities increased to control levels or higher.The activity of glycogen synthase phosphatase was not decreased in diabetic skeletal muscle. This observation, coupled with the rapid response of the diabetic synthase to in vivo insulin treatment, suggests that, unlike the phosphatase in cardiac muscle and liver, the glycogen synthase phosphatase in skeletal muscle is not altered by the diabetic state.Abbreviations UDPG uridine diphosphoglucose - G6P glucose 6-phosphate - EDTA ethylene diamine tetraacetic acid - IP intraperitoneally - MOPS morpholinopropane sulfonic acid - -ME -mercaptoethanol - V_G6P calculated velocity of the enzyme in the presence of infinite G6P concentration - V_UDPG calculated velocity of the enzyme in the presence of infinite UDPG concentration     

Glucosamine and glucose induce insulin resistance by different mechanisms in rat skeletal muscle

It has been hypothesized that glucose-induced insulin resistance is mediated by accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs). In a previous study on rat epitrochlearis muscles incubated with high concentrations of glucose and insulin (Kawanaka K, D-H Han, J Gao, LA Nolte, and JO Holloszy. J Biol Chem 276: 20101-20107, 2001), we found that insulin resistance developed even when the increase in UDP-Hex-NAcs was prevented. Furthermore, actinomycin D completely prevented glucose-induced insulin resistance despite a greater accumulation of UDP-HexNAcs. In the present study, we used the same epitrochlearis muscle preparation, as well as the rat hemidiaphragm, to determine whether, like glucose, glucosamine causes insulin resistance by an actinomycin D-inhibitable process. Incubation of diaphragm muscles with 10 mM glucosamine for 3 h resulted in an approximately fivefold increase in UDP-HexNAcs, an approximately 50% reduction in insulin responsiveness of glucose transport, and a 58% reduction in ATP concentration. These effects of glucosamine were not prevented by actinomycin D. Incubation of epitrochlearis muscles with 20 mM glucosamine for 3 h or with 10 mM glucosamine for 5 h also caused large decreases in insulin responsiveness of glucose transport but with no reduction in ATP concentration. Actinomycin D did not prevent the glucosamine-induced insulin resistance. The insulin-induced increases in tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the binding of PI 3-kinase to IRS-1 were decreased approximately 60% in epitrochlearis muscles exposed to glucosamine. This is in contrast to glucose-induced insulin resistance, which was not associated with impaired insulin signaling. These results provide evidence that glucosamine and glucose induce insulin resistance by different mechanisms.     

Levonorgestrel and norethindrone alter insulin action on skeletal muscle of the female rat

Prospective studies of women receiving oral contraceptives suggest that the progestin component may induce insulin resistance and variable deterioration of glucose tolerance. Because the tissue sites and nature of this insulin antagonism are not well-defined, we studied the effects of two parenterally administered progestins, levonorgestrel (NG) and norethindrone (NE), on insulin-regulated glucose uptake and phenylalanine release by the perfused rat hindquarter. Female rats were injected sc for 14 days with NG or NE (10 or 30 micrograms/kg/day). Low-dose NG and high-dose NE approximate the per kg dose received by women taking a high-dose progestin oral contraceptive. Phenylalanine release and glucose uptake (nmole/min/g) by the perfused hindquarters were calculated from the A-V difference for each. Progestin treatment (30 micrograms/kg/d) significantly reduced phenylalanine release from hindquarters perfused without exogenous insulin. Hindquarters from the high dose NG and low and high dose NE rats perfused with insulin (100 microU/ml) released 22% less phenylalanine than control rats perfused with the same insulin concentration (P less than 0.01) but the net suppression below baseline was similar in the control and steroid-treated groups. High-dose progestin treatment did not alter glucose uptake by hindquarters perfused without exogenous insulin. Insulin (100 microU/ml) increased glucose uptake by hindquarters of control and progestin-treated rats as compared to animals in the same treatment group perfused without exogenous insulin (P less than 0.01). High dose NE impaired insulin-stimulated glucose uptake 24% below values of the control group (P less than 0.01). The other NE and NG doses had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)