Methylphenidate for gait impairment in Parkinson disease: a randomized clinical trial.

BACKGROUND: There is a paucity of therapies for gait impairment in Parkinson disease (PD). Open-label studies have suggested improved gait after treatment with methylphenidate (MPD). OBJECTIVE: To evaluate the efficacy of MPD for the treatment of gait impairment in PD. METHODS: Twenty-seven subjects with PD and moderate gait impairment were screened for this 6-month placebo-controlled, double-blind study. Subjects were randomly assigned to MPD (maximum, up to 80 mg/day) or placebo for 12 weeks and crossed over after a 3-week washout. The primary outcome measure was change in a gait composite score (stride length + velocity) between groups at 4 and 12 weeks. Secondary outcome measures included changes in motor function, as measured by the Unified Parkinson's Disease Rating Scale (UPDRS), Freezing of Gait Questionnaire (FOGQ), number of gait-diary freezing episodes, and measures of depression, sleepiness, and quality of life. Three-factor repeated-measures analysis of variance was used to measure changes between groups. RESULTS: Twenty-three eligible subjects with PD were randomized and 17 completed the trial. There was no change in the gait composite score or treatment or time effect for any of the variables. Treatment effect was not modified by state or study visit. Although there was a trend for reduced frequency of freezing and shuffling per diary, the FOGQ and UPDRS scores worsened in the MPD group compared to placebo. There was a marginal improvement in some measures of depression. CONCLUSIONS: MPD did not improve gait and tended to worsen measures of motor function, sleepiness, and quality of life. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence for the lack of benefit of MPD on PD-associated gait impairment. CLINICAL TRIAL REGISTRATION: NCT00526630.

Small molecule insulin receptor activators potentiate insulin action in insulin-resistant cells.

In type 2 diabetes, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. To study a new class of antidiabetic agents, we compared two small, nonpeptide molecules that activate insulin receptor (IR) beta-subunit tyrosine kinase activity: Merck L7, a direct IR agonist, and Telik's TLK16998, an IR sensitizer. In rat hepatoma cells (HTCs) that overexpress the IR (HTC-IR), IR autophosphorylation was directly activated by L7 in the absence of insulin. TLK16998 did not directly activate IR autophosphorylation, but it enhanced IR autophosphorylation in the presence of insulin. Tyrosine phosphorylation of an endogenous 185-kDa IR substrate was also significantly enhanced by both Merck L7 alone and TLK16998 plus insulin. Adding TLK16998 to L7 produced synergistic effects, further indicating that these two compounds act on the IR through separate mechanisms. We next studied HTC-IR(Delta485-599) cells, which overexpress a mutant IR with a deletion in the alpha-subunit connecting domain that does not undergo autophosphorylation in response to insulin binding. L7 was able to directly activate autophosphorylation of the deletion mutant IR in these cells, whereas TLK16998 had no effect. Compounds were then tested in three other cell models of impaired IR function. Both TLK16998 and Merck L7 improved IR autophosphorylation in cells with diminished IR signaling due to either treatment with tumor necrosis factor-alpha or overexpression of membrane glycoprotein PC-1. However, in TPA (tetradecanoylphorbol acetate)-treated cells, TLK16998 but not Merck L7 was able to significantly reverse the impaired insulin-stimulated IR autophosphorylation. In summary, these two classes of IR activators selectively increased IR function in a variety of insulin-resistant cell lines.

Protection against oxidative stress-induced insulin resistance in rat L6 muscle cells by mircomolar concentrations of alpha-lipoic acid.

In diabetic patients, alpha-lipoic acid (LA) improves skeletal muscle glucose transport, resulting in increased glucose disposal; however, the molecular mechanism of action of LA is presently unknown. We studied the effects of LA on basal and insulin-stimulated glucose transport in cultured rat L6 muscle cells that overexpress GLUT4. When 2-deoxy-D-glucose uptake was measured in these cells, they were more sensitive and responsive to insulin than wild-type L6 cells. LA, at concentrations < or = 1 mmol/l, had only small effects on glucose transport in cells not exposed to oxidative stress. When cells were exposed to glucose oxidase and glucose to generate H2O2 and cause oxidative stress, there was a marked decrease in insulin-stimulated glucose transport. Pretreatment with LA over the concentration range of 10-1,000 pmol/l protected the insulin effect from inhibition by H2O2. Both the R and S isomers of LA were equally effective. In addition, oxidative stress caused a significant decrease (approximately 50%) in reduced glutathione concentration, along with the rapid activation of the stress-sensitive p38 mitogen-activated protein kinase. Pretreatment with LA prevented both of these events, coincident with protecting insulin action. These studies indicate that in muscle, the major site of insulin-stimulated glucose disposal, one important effect of LA on the insulin-signaling cascade is to protect cells from oxidative stress-induced insulin resistance.

PC-1 nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification.

Inogranic pyrophosphate (PPi) inhibits hydroxyapatite deposition, and mice deficient in the PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) Plasma cell membrane glycoprotein-1 (PC-1) develop peri-articular and arterial calcification in early life. In idiopathic infantile arterial calcification (IIAC), hydroxyapatite deposition and smooth muscle cell (SMC) proliferation occur, sometimes associated with peri-articular calcification. Thus, we assessed PC-1 expression and PPi metabolism in a 25-month-old boy with IIAC and peri-articular calcifications. Plasma PC-1 was <1 ng/ml by enzyme-linked immunosorbent assay in the proband, but 10 to 30 ng/ml in unaffected family members and controls. PC-1 functioned to raise extracellular PPi in cultured aortic SMCs. However, PC-1 was sparse in temporal artery lesion SMCs in the proband, unlike the case for SMCs in atherosclerotic carotid artery lesions of unrelated adults. Proband plasma and explant-cultured dermal fibroblast NTPPPH and PPi were markedly decreased. The proband was heterozygous at the PC-1 locus, and sizes of PC-1 mRNA and polypeptide, and the PC-1 mRNA-coding region sequence were normal in proband fibroblasts. However, immunoreactive PC-1 protein was relatively sparse in proband fibroblasts. In conclusion, deficient extracellular PPi and a deficiency of PC-1 NTPPPH activity can be associated with human infantile arterial and peri-articular calcification, and may help explain the sharing of certain phenotypic features between some IIAC patients and PC-1-deficient mice.

Membrane glycoprotein PC-1 inhibition of insulin receptor function occurs via direct interaction with the receptor alpha-subunit.

Plasma cell membrane glycoprotein-1 (PC-1) inhibits insulin receptor (IR) tyrosine kinase activity and subsequent cellular signaling. PC-1 content is elevated in fibroblasts, muscle, and adipose tissue from insulin-resistant subjects, and its elevation correlates with in vivo insulin resistance. In vitro, when PC-1 is transfected and overexpressed in cultured cells, it inhibits IR tyrosine kinase activity. To determine the mechanism whereby PC-1 regulates the IR, we studied how PC-1 interacts with this protein. Overexpression of PC-1 in MCF-7 cells inhibited tyrosine kinase activity of the IR, but not of the IGF-I receptor. When the IR was immunocaptured by specific IR monoclonal antibodies, PC-1 was associated with this receptor. In contrast, after specific immunocapture, PC-1 was not associated with the IGF-I receptor. We next studied HTC cells that were overexpressing an IR alpha-subunit mutant. This IR mutant binds insulin but has a deletion in the tyrosine kinase regulatory domain located in amino acids 485-599. In contrast to normal IRs, PC-1 did not associate with this mutant and did not affect tyrosine kinase activity. To determine whether decreasing PC-1 expression would reverse the inhibition of tyrosine kinase activity, we treated MCF-7 cells overexpressing PC-1 with a monoclonal antibody to PC-1. This treatment decreased PC-1 levels; concomitantly, IR tyrosine kinase activity increased. In contrast, IGF-I receptor tyrosine kinase activity was not increased. These studies indicate, therefore, that PC-1 may inhibit the IR by interacting directly with a specific region in the IR alpha-subunit. These studies also raise the possibility that monoclonal antibodies to PC-1 could be a new treatment for insulin resistance.

Overexpression of membrane glycoprotein PC-1 in MDA-MB231 breast cancer cells is associated with inhibition of insulin receptor tyrosine kinase activity.

MDA-MB231 human breast cancer cells are unresponsive to insulin and contain a glycoprotein inhibitor of insulin-stimulated insulin receptor (IR) tyrosine kinase activity. Prior studies in both fibroblasts from insulin- resistant non-insulin-dependent diabetes mellitus patients and transfected cells indicate that overexpression of membrane glycoprotein PC-1 reduces IR tyrosine kinase activity. In the present study, we measured PC-1 content and activity in MDA-MB231 and four other human breast cancer cell lines. We observed that PC-1 expression was 3- to 30-fold higher in MDA-MB231 cells when compared with the other breast cell lines. Wheat germ agglutinin extracts of MDA-MB231 cells inhibited IR tyrosine kinase activity. Treatment of these extracts with an antibody to PC-1 significantly reduced their ability to inhibit insulin-stimulated IR tyrosine kinase activity. In addition, when cell clones with different PC-1 activity were selected from MDA-MB231 cells, we found an inverse correlation (r = -0.741, P = 0.006) between the PC-1 activity and the insulin-stimulated IR autophosphorylation. A similar inverse correlation was observed in cell clones derived from the insulin-responsive breast cancer cell line MCF-7. By both immunoprecipitation and cross-linking studies we found PC-1 to be associated with IR. These studies indicate, therefore, that overexpression of PC-1 in MDA-MB231 cells may account, at least in part, for the reduced IR tyrosine kinase activity and suggest that PC-1 is a specific modulator of the IR activity in breast cancer cells.

Skeletal muscle content of membrane glycoprotein PC-1 in obesity. Relationship to muscle glucose transport.

Membrane glycoprotein PC-1, an inhibitor of insulin signaling, produces insulin resistance when overexpressed in cells transfected with PC-1 cDNA. In the present study, we determined whether PC-1 plays a role in the insulin resistance of skeletal muscle in obesity. Rectus abdominus muscle biopsies were taken from patients undergoing elective surgery. Subjects included both NIDDM patients (n = 14) and nondiabetic patients (n = 34) across a wide range of BMI values (19.5-90.1). Insulin-stimulated glucose transport was measured in incubated muscle strips, and PC-1 content, enzymatic activity, and insulin receptor content were measured in solubilized muscle extracts. Increasing BMI correlated with both an increase in the content of PC-1 in muscle (r = 0.55, P < 0.001) and a decrease in insulin stimulation of muscle glucose transport (r = -0.58, P = 0.008). NIDDM had no effect on either PC-1 content or glucose transport for any given level of obesity. Insulin stimulation of muscle glucose transport was negatively related to muscle PC-1 content (r = -0.68, P = 0.001) and positively related to insulin receptor content (r = 0.60, P = 0.005). Multivariate analysis indicated that both skeletal muscle PC-1 content and insulin receptor content, but not BMI, were independent predictors of insulin-stimulated glucose transport. Muscle PC-1 content accounted for 42% and insulin receptor content for 17% of the variance in glucose transport values. These studies raise the possibility that increased expression of PC-1 and a decreased insulin receptor content in skeletal muscle may be involved in the insulin resistance of obesity.

Membrane glycoprotein PC-1 and insulin resistance in non-insulin-dependent diabetes mellitus.

Most patients with non-insulin-dependent diabetes mellitus are resistant to both endogenous and exogenous insulin. Insulin resistance precedes the onset of this disease, suggesting that it may be an initial abnormality. Insulin-receptor kinase activity is impaired in muscle, fibroblasts and other tissues of many patients with non-insulin-dependent diabetes mellitus, but abnormalities in the insulin-receptor gene do not appear to be the cause of this decreased kinase activity. Skin fibroblasts from certain insulin-resistant patients contain an inhibitor of insulin-receptor tyrosine kinase. Here we show that this inhibitor is a membrane glycoprotein, termed PC-1 (refs 10, 11). We find that PC-1 activity is increased in fibroblasts from seven of nine patients with typical non-insulin-dependent diabetes mellitus. In addition, overexpression of PC-1 in transfected cultured cells reduces insulin-stimulated tyrosine kinase activity. These studies raise the possibility that PC-1 has a role in the insulin resistance of non-insulin-dependent diabetes mellitus.

High-affinity insulin binding to an atypical insulin-like growth factor-I receptor in human breast cancer cells.

We studied the nature of insulin receptor binding in MCF-7 breast cancer cells. In both intact cells and solubilized receptor preparations, high-affinity insulin binding was seen. However, unlabeled insulin-like growth factor-I (IGF-I) was five-fold more potent in inhibiting 125I-insulin binding than insulin itself. With monoclonal antibodies to the insulin receptor, 30% of 125I-insulin binding was inhibited. In contrast when alpha-IR3, a monoclonal antibody that recognizes typical IGF-I receptor, was employed over 60% of 125I-insulin binding was inhibited. The B29-MAB-125I-insulin photoprobe was then cross-linked to MCF-7 membranes. Cross-linking was inhibited by both unlabeled insulin and IGF-I. Further, the B29-MAB-125I-insulin photoprobe cross-linked to MCF-7 membranes was strongly immunoprecipitated by alpha-IR3. Employing sequential affinity chromatography with insulin-Affi-gel followed by insulin receptor monoclonal antibody agarose, atypical insulin binding activity was separated from insulin receptor binding activity. This atypical receptor had intrinsic tyrosine kinase activity. Both insulin and IGF-I stimulated the phosphorylation of the receptor's beta subunit. In MCF-7 cells both IGF-I and insulin stimulated [3H]thymidine incorporation; alpha-IR3 blocked all of the IGF-I effect but only 50-60% of the insulin effect. This study demonstrates in MCF-7 cells that, in addition to typical insulin and IGF-I receptors, there is another receptor that binds both insulin and IGF-I with high affinity.

Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163.

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.

Progestins induce down-regulation of insulin-like growth factor-I (IGF-I) receptors in human breast cancer cells: potential autocrine role of IGF-II.

Insulin-like growth factor-I (IGF-I) receptors are present in breast cancer cells and may play a role in breast cancer cell growth. We have studied the effect of progestins on IGF-I receptors in T47D human breast cancer cells. T47D cells constitutively express high levels of progesterone receptors and are a model for studying the regulation of cellular functions by progestins. Treatment of T47D cells with either progesterone or the synthetic progestin promegestone (R5020) decreased IGF-I receptor content by approximately 50%, as measured by Scatchard analysis and receptor biosynthesis studies. In contrast to progestins, estradiol, dexamethasone, and dihydrotestosterone did not influence IGF-I receptor content. No effect of R5020 was seen after 12 h of incubation, a near-maximal effect was seen after 24 h, and greatest effects were seen after 72 h. R5020 decreased IGF-I receptor mRNA abundance, indicating that progestins acted at the level of gene expression. However, progestins also increased the secretion of IGF-II, a ligand for the IGF-I receptor. In contrast to IGF-II, T47D cells did not express IGF-I. The addition of exogenous IGF-II to T47D cells down-regulated both IGF-I receptor binding and IGF-I receptor mRNA abundance. This study indicates, therefore, that progestins regulate IGF-I receptors in breast cancer cells and suggests that this regulation occurs via an autocrine pathway involving enhanced IGF-II secretion.

Evidence that insulin plus ATP may induce a conformational change in the beta subunit of the insulin receptor without inducing receptor autophosphorylation.

The effect of insulin and ATP on insulin receptor beta subunit conformation was studied in vitro with radioiodinated monoclonal antibodies directed at several regions of the receptor beta subunit. Insulin plus ATP inhibited their binding to the receptor. The greatest inhibitory effect of insulin and ATP was seen with antibody 17A3 which recognizes a domain of the beta subunit that is near the major tyrosine autophosphorylation sites at residues 1158, 1162, and 1163. ATP alone inhibited 17A3 binding with a one-half maximal ATP inhibitory concentration of 186 +/- 7 microM. Insulin at concentrations as low as 100 pM potentiated the effect of ATP; at 100 nM where insulin had its maximal effect, insulin lowered the one-half maximal inhibitory concentration of ATP to 16 +/- 6 microM. At 1 mM CTP, GTP, ITP, TTP, and AMP were without effect in either the presence or absence of insulin; in contrast, ADP was inhibitory in the presence of insulin. Of major interest was adenyl-5'-yl imidodiphosphate (AMP-PNP). This nonhydrolyzable analog of ATP inhibited 17A3 binding, and the effect of AMP-PNP (like ATP) was potentiated by insulin. Two insulin receptor beta subunit mutants then were studied. Mutant receptor F3, where the major tyrosine autophosphorylation sites at residues 1158, 1162, and 1163 were changed to phenylalanines, bound to 17A3; antibody binding was inhibited by insulin and ATP in a manner similar to normal receptors. In contrast, mutant receptor M1030, where the lysine in the ATP binding site at residue 1030 was changed to methionine, bound 17A3, but unlike either normal receptors or F3 receptors, the binding of 17A3 was not inhibited by insulin and ATP. Therefore, these studies raise the possibility that, in vivo, ATP binding in the presence of insulin may induce a conformational change in the insulin receptor beta subunit which in turn signals some of the biological effects of insulin.

Overexpression of insulin receptors in fibroblast and ovary cells induces a ligand-mediated transformed phenotype.

To investigate whether overexpression of the insulin receptor results in altered cell growth we used NIH 3T3 cells transfected with a bovine papilloma virus/insulin receptor cDNA construct (3T3/HIR). These cells expressed high numbers of insulin receptors (mean +/- sd, 631.0 +/- 16.7 ng receptors/10(6) cells). Insulin significantly stimulated the growth of 3T3/HIR cells maintained in serum-free medium. Moreover, in these cells, insulin induced marked phenotypic changes, including alterations in cell shape, loss of contact inhibition, and focal growth. In contrast to 3T3/HIR cells, insulin was without effect in either wild-type 3T3 cells (3T3/wt), 3T3 cells transfected with the neomycin resistance gene (3T3/NEO), or the bovine papilloma virus (3T3/BPV). To assess the presence of anchorage-independent growth, cells were seeded in soft agar and inspected for colony formation. 3T3/HIR cells showed absent or minimal colony growth in the absence of insulin. However, there was a dose-dependent insulin-stimulated increase in both colony size and number. Insulin-stimulated colony formation was specifically inhibited by an insulin antagonist, monoclonal antibody MA-10. In the presence of 100 nM insulin, about 3% of cells formed large colonies. Insulin neither stimulated growth nor induced colony formation in 3T3/wt cells or 3T3/NEO cells. Insulin also stimulated colony formation in CHO cells transfected with an insulin receptor cDNA construct. In conclusion, overexpression of normal insulin receptors induces a ligand-dependent transformed phenotype. This phenomenon may have clinical relevance by conferring a selective growth advantage to tumor cells with high numbers of insulin receptors.

Production of inhibitor of insulin-receptor tyrosine kinase in fibroblasts from patient with insulin resistance and NIDDM.

Although non-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in insulin action, the molecular basis of this resistance is unknown. We studied fibroblasts from a markedly insulin-resistant patient with NIDDM but without acanthosis nigricans. Her fibroblasts were resistant to insulin when alpha-aminoisobutyric acid uptake was measured. Fibroblasts from this patient demonstrated normal insulin-receptor content as measured by both insulin-receptor radioimmunoassay and by Scatchard analysis. However, when compared with nondiabetic control subjects, insulin-receptor kinase assays of wheat-germ-purified receptors prepared from her fibroblasts showed very low basal and no insulin-stimulated tyrosine kinase activity. The insulin receptor was then removed from the wheat-germ fraction by monoclonal antibody affinity chromatography. This insulin-receptor-deficient fraction inhibited both basal and insulin-stimulated tyrosine kinase activity of highly purified insulin receptors. When the specificity of this inhibition was tested, less inhibition was seen with insulinlike growth factor I-receptor tyrosine kinase, and even less inhibition was seen with the proto-oncogene p60c-src tyrosine kinase. Thus, these studies indicate that fibroblasts from an insulin-resistant patient with NIDDM produce a relatively specific glycoprotein inhibitor of insulin-receptor tyrosine kinase. Therefore, these studies raise the possibility that this inhibitor may play an important role in the insulin resistance seen in this patient.

Monoclonal antibodies mimic insulin activation of ribosomal protein S6 kinase without activation of insulin receptor tyrosine kinase. Studies in cells transfected with normal and mutant human insulin receptors.

The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.

Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase.

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell. In these cells, as well as in nontransfected cells, insulin stimulated the uptake of alpha-aminoisobutyric acid. Two monoclonal antibodies directed against the human insulin receptor alpha subunit, like insulin, stimulated amino acid uptake in transfected HTC cells, but not in nontransfected HTC cells. The antibodies, in contrast to insulin, failed to stimulate insulin receptor tyrosine kinase activity, both in intact transfected cells and in cell free extracts prepared from them. These data suggest, therefore, that activation of insulin receptor tyrosine kinase may not be an obligatory step in all of the transmembrane signaling mechanisms of the insulin receptor.

Mechanisms of insulin-induced insulin-receptor downregulation. Decrease of receptor biosynthesis and mRNA levels.

The influence of insulin on the downregulation of its receptor was studied in AR42J cultured pancreatic acinar cells, a cell line that has been demonstrated to be metabolically responsive to insulin. Downregulation induced by insulin was time and dose dependent. After a 20-h incubation with 1 microM insulin, Scatchard analysis revealed approximately 80% loss of insulin receptors. Studies of receptor half-life indicated that treatment with insulin accelerated the degradation of both the alpha- and beta-subunits of the insulin receptor by 30-60%. In addition, biosynthetic-labeling studies indicated that insulin inhibited the biosynthesis of the insulin-receptor precursor by greater than 30%. This decreased biosynthesis of the precursor was associated with decreased production of mature receptor subunits. Poly(A)+ RNA was extracted from control cells and cells treated for 24 h with 100 nM insulin. Slot blots and Northern transfers revealed that insulin induced an approximately 50% decrease in insulin-receptor mRNA levels. Therefore, these studies indicate that insulin may diminish the concentration of its receptors in target cells by at least two mechanisms: acceleration of receptor degradation and inhibition of receptor biosynthesis at the level of mRNA.

Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor alpha-subunit.

Affinity-purified insulin receptor was photoaffinity labeled with a cleavable radioactive insulin photoprobe. Exhaustive digestion of the labeled alpha-subunit with endoproteinase Glu-C produced a major radioactive fragment of 23 kDa as a part of the putative insulin-binding domain. This fragment could contain either residues 205-316 or 518-633 of the alpha-subunit. Rat hepatoma cells and Chinese hamster ovary cells were transfected with cDNA encoding a human insulin receptor mutant with a deletion of the cysteine-rich region spanning amino acid residues 124-319. Insulin binding by these cells was not increased in spite of high numbers of the mutant insulin receptors being expressed. A panel of monoclonal antibodies which was specific for the receptor alpha-subunit and inhibited insulin binding immunoprecipitated the photolabeled 23-kDa receptor fragment but not the receptor mutant. A synthetic peptide containing residues 243-251 was specifically bound by agarose-insulin beads. We therefore suggest that the 23-kDa fragment contains residues 205-316, and that insulin binding occurs, in part, in the cysteine-rich region of the alpha-subunit.

Insulin and insulin-like growth factor I regulate the same biological functions in HEP-G2 cells via their own specific receptors.

The receptors for insulin and insulin-like growth factor I (IGF-I) are closely related molecules, with an extracellular binding domain and an intracellular tyrosine kinase domain. The interaction of insulin and IGF-I with their respective receptors activates the receptor kinase domain, leading to the biological actions of the hormones. Since insulin generally regulates metabolic events and IGF-I generally regulates growth events, it is believed that structural differences in the tyrosine kinase domains of the two respective receptors may elicit different biological responses via different transmembrane signaling mechanisms. We studied the regulation of glycogen metabolism and amino acid uptake in human cultured HEP-G2 hepatoma cells, which have distinct receptors for both insulin and IGF-I. The receptor specificity of these responses was probed with specific monoclonal antibodies to both the insulin and IGF-I receptors. Stimulation of both [3H]glucose incorporation into glycogen and alpha-[3H]aminoisobutyric acid uptake by insulin was half-maximal at concentrations of 1-5 nmol/L. These effects were blocked by the insulin receptor monoclonal antibody MA-10, but not by the IGF-I receptor antibody alpha IR-3. Stimulation of both functions by IGF-I was half-maximal at concentrations of 1-5 nmol/L, and these effects were inhibited by alpha IR-3, but not by MA-10. These studies indicate that in HEP-G2 cells both insulin and IGF-I, via their own receptors, stimulate the same biological responses.

Mechanism of glucocorticoid receptor down-regulation by glucocorticoids.

The effect of glucocorticoids on the regulation of glucocorticoid receptor mRNA was studied in two different cell lines, human IM-9 lymphocytes and rat pancreatic acinar AR42J cells. Using a glucocorticoid receptor cDNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and quantitated by slot-blot hybridization. In IM-9 and AR42J cells, dexamethasone decreased steady-state glucocorticoid receptor mRNA levels to approximately 50% of control. This decrease occurred with a one-half time of 3 h for IM-9 cells and 6 h for AR42J cells. Dexamethasone was the most potent steroid tested with a one-half maximal effect occurring at 10 nM and a maximal effect occurring at 100 nM. Glucocorticoid receptor mRNA half-life and gene transcription were then studied to determine the mechanism of decreased mRNA levels. The glucocorticoid mRNA half-life was approximately 120 min in IM-9 cells and 240 min in AR42J cells; these rates were not affected by dexamethasone treatment. In contrast, the rate of glucocorticoid gene transcription as measured by run-on assays in IM-9 cells was decreased to 50 +/- 6% of control by dexamethasone. These results indicate therefore that glucocorticoids regulate glucocorticoid receptor mRNA levels by influencing gene transcription.