Search PubMed⌕ Search

Biomedical subjects

M J Quon

Publications and source records attributed to M J Quon.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effects of overexpressing wild-type and mutant PDGF receptors on translocation of GLUT4 in transfected rat adipose cells.

Activation of phosphatidylinositol 3-kinase (PI3K) by insulin is necessary for the effect of insulin to recruit GLUT4 to the cell surface in insulin target cells. In adipose cells, stimulation of endogenous PDGF receptors (PDGF-R) results in increased PI3K activity without causing recruitment of GLUT4. We overexpressed wild-type or mutant forms of the PDGF-R in rat adipose cells and examined their effects on PDGF- and insulin-stimulated recruitment of co-transfected epitope-tagged GLUT4. Control cells expressing only tagged GLUT4 had a 3-fold increase in cell surface GLUT4 upon insulin stimulation but no response to PDGF. Cells overexpressing wild-type PDGF-R maintained insulin responsiveness and, in addition, acquired the ability to recruit GLUT4 in response to PDGF. Surprisingly, overexpression of F740/ F751 (mutant PDGF-R unable to directly activate PI3K) led to similar results. Nevertheless, wortmannin (an inhibitor of PI3K) blocked effects of both PDGF and insulin to recruit GLUT4. Our data suggest that overexpression of PDGF-R mediates positive effects on GLUT4 translocation by a wortmannin sensitive pathway not dependent on direct interaction of the PDGF-R with PI3K.

Adipose Tissue↗
PubMed1996

Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells.

Hypertension is associated with insulin-resistant states such as diabetes and obesity. Nitric oxide (NO) contributes to regulation of blood pressure. To gain insight into potential mechanisms linking hypertension with insulin resistance we directly measured and characterized NO production from human umbilical vein endothelial cells (HUVEC) in response to insulin using an amperometric NO-selective electrode. Insulin stimulation of HUVEC resulted in rapid, dose-dependent production of NO with a maximal response of approximately 100 nM NO (200,000 cells in 2 ml media; ED50 approximately 500 nM insulin). Although HUVEC have many more IGF-1 receptors than insulin receptors (approximately 400,000, and approximately 40,000 per cell respectively), a maximally stimulating dose of IGF-1 generated a smaller response than insulin (40 nM NO; ED50 approximately 100 nM IGF-1). Stimulation of HUVEC with PDGF did not result in measurable NO production. The effects of insulin and IGF-1 were completely blocked by inhibitors of either tyrosine kinase (genestein) or nitric oxide synthase (L-NAME). Wortmannin (an inhibitor of phosphatidylinositol 3-kinase [PI 3-kinase]) inhibited insulin-stimulated production of NO by approximately 50%. Since PI 3-kinase activity is required for insulin-stimulated glucose transport, our data suggest that NO is a novel effector of insulin signaling pathways that are also involved with glucose metabolism.

Androstadienes↗
PubMed1996

Characterization of a mutant GLUT4 lacking the N-glycosylation site: studies in transfected rat adipose cells.

GLUT4, the insulin-responsive glucose transporter expressed primarily in muscle and adipose tissue, contains a single N-glycosylation site. We characterized a mutant GLUT4 lacking the N-glycosylation site (Asn57-->Gln) in primary cultures of rat adipose cells. We transiently transfected cells with expression vectors for epitope-tagged GLUT4 containing either wild-type (GLUT4-HA) or mutant (GLN57-HA) cDNA sequences. Expression of GLN57-HA in adipose cells was approximately 10-fold lower than for GLUT4-HA even though mRNA levels for both recombinant transporters were comparable. Biosynthetic labeling studies showed markedly decreased incorporation of [35S]-methionine/cysteine into GLN57-HA relative to GLUT4-HA consistent with either a decreased synthetic rate or accelerated degradation of GLN57-HA. Interestingly, transient transfection of GLUT4-HA and GLN57-HA in COS-7 cells (which do not express endogenous GLUT4) resulted in comparable levels of protein expression for both transporters. Thus, in the physiologically relevant adipose cell, glycosylation of GLUT4 appears to play an important functional role.

Adipose Tissue↗
PubMed1995

The mouse obese gene. Genomic organization, promoter activity, and activation by CCAAT/enhancer-binding protein alpha.

The obese gene product, leptin, regulates adiposity. Mice homozygous for a nonfunctional obese gene become massively obese and develop diabetes mellitus due to overeating and increased metabolic efficiency. The cDNA sequence of obese was recently reported (Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J. L. (1994) Nature 372, 425-432; Correction: (1995 Nature 374, 479). We have determined the genomic organization of the 5' end of the mouse obese gene. The coding sequence is in exons 2 and 3. A single TATA-containing promoter was found upstream of exon 1. A minority (probably approximately 5%) of the obese mRNA contained an extra, untranslated exon between exons 1 and 2. Transcription of the obese gene was detected only in adipose cells. A 762-base pair obese gene promoter driving a luciferase gene yielded abundant activity in transiently transfected rat adipose cells in primary culture. The obese promoter was inactive in erythroid K562 cells. Deletion of bases from -762 downstream to -161 did not affect promoter activity in transfected adipose cells. The -161 minimal promoter contained consensus Sp1 and CCAAT/enhancer-binding protein (C/EBP) motifs. Cotransfection with C/EBP alpha (a transcription factor important in adipose cell differentiation) caused 23-fold activation. These data suggest that the obese promoter is a natural target of C/EBP alpha.

Adipose Tissue↗
PubMed1995

Two mutant alleles of the insulin receptor gene in a family with a genetic form of insulin resistance: a 10 base pair deletion in exon 1 and a mutation substituting serine for asparagine-462.

Mutations in the insulin receptor gene cause several genetic syndromes associated with extreme insulin resistance. We have studied three insulin resistant siblings with acanthosis nigricans, dental abnormalities, and acral hypertrophy. The female patient also had primary amenorrhea due to hyperandrogenism. All three patients were compound heterozygotes with two mutant alleles of the insulin receptor gene. One allele had a 10-bp deletion in the region of exon 1 encoding the hydrophobic signal peptide; this leads to a frameshift and premature chain termination at codon 61. The deletion occurs at the site of a direct repeat of a hexanucleotide sequence interrupted by a tetranucleotide sequence; the deletion may have resulted from recombination between the upstream and downstream hexanucleotide repeats. In the other mutant allele, there is a missense mutation substituting serine for Asn462-a mutation identified previously in one allele of the insulin receptor gene in a patient with type-A insulin resistance. The Ser462 mutation impaired the ability of acidic pH to dissociate insulin from the receptor. Thus, Thus, like the previously described Glu460 mutation, the Ser462 mutation may retard dissociation of insulin from the receptor in the acidic compartment of the endosome and may, as a result, accelerate the rate of receptor degradation.

Alleles↗
PubMed1995

Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.

Adipocytes↗
PubMed1994

Insulin receptor substrate 1 mediates the stimulatory effect of insulin on GLUT4 translocation in transfected rat adipose cells.

Insulin signaling is initiated at least in part by activation of the insulin receptor tyrosine kinase and subsequent phosphorylation of cellular substrates such as insulin receptor substrate 1 (IRS-1). Previous studies have focused on the role of IRS-1 in the mitogenic actions of insulin. We have now investigated the possible role of IRS-1 in mediating the effect of insulin to stimulate glucose transport in a physiologically relevant insulin target tissue. In this study, we transfected rat adipose cells in primary culture with an antisense ribozyme directed against rat IRS-1. Expression of the ribozyme in these cells caused a 4.4-fold increase in the concentration of insulin required to achieve half-maximal stimulation of the translocation of cotransfected epitope-tagged GLUT4 without changing the maximal insulin response. Overexpression of human IRS-1 increased the basal cell surface GLUT4 to nearly the maximal level in the absence of insulin. When the ribozyme (specific to rat IRS-1) was cotransfected along with human IRS-1, the insulin dose-response curve was shifted to the left when compared with cells transfected with the ribozyme alone. These data provide strong support for the hypothesis that IRS-1 plays a role in insulin-stimulated glucose transport in insulin-responsive cells.

Adipose Tissue↗
PubMed1994

Tyrosine kinase-deficient mutant human insulin receptors (Met1153-->Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4.

Insulin regulates essential pathways for growth, differentiation, and metabolism in vivo. We report a physiologically relevant system for dissecting the molecular mechanisms of insulin signal transduction related to glucose transport. This is an extension of our recently reported method for transfection of DNA into rat adipose cells in primary culture. In the present work, cDNA coding for GLUT4 with an epitope tag (HA1) in the first exofacial loop is used as a reporter gene so that GLUT4 translocation can be studied exclusively in transfected cells. Insulin stimulates a 4.3-fold recruitment of transfected epitope-tagged GLUT4 to the cell surface. Cells cotransfected with the reporter gene and the human insulin receptor gene show an increase in cell surface GLUT4 in the basal state (no insulin) to levels comparable to those seen with maximal insulin stimulation of cells transfected with the reporter gene alone. In contrast, cells overexpressing a naturally occurring tyrosine kinase-deficient mutant insulin receptor (Met1153-->Ile) show no increase in the basal cell surface GLUT4 and no shift in the insulin dose-response curve relative to cells transfected with the reporter gene alone. These results demonstrate that insulin receptor tyrosine kinase activity is essential in insulin-stimulated glucose transport in adipose cells.

Adipose Tissue↗
PubMed1994

Mechanisms of hormone resistance: lessons from insulin-resistant patients.

Hormones are secreted by endocrine glands and transported to the target cell at which the hormone acts. The hormone binds to its receptor, thereby eliciting various biological responses within the target cell. Examples of disease mechanisms that function at the different stages in the development of the insulin receptor, and result in insulin resistance, are discussed in this review. Antibodies to insulin can impair delivery of the hormone to the target cell, and can desensitize that target cell to insulin action. In recent years, several genetic diseases have been identified that result from mutations in the genes encoding the relevant receptors. Studies of syndromes of insulin resistance provide illustrations of the multiple types of defects in receptor function that can generally cause hormone resistance (12, 13). For example, mutations in the receptor can decrease the number of receptors on the cell surface by inhibiting receptor biosynthesis, impairing receptor transport to the cell surface, or accelerating the rate of receptor degradation. Alternatively, mutations have been identified that decrease the affinity of insulin binding or inhibit receptor tyrosine kinase activity. In recent years, there has been considerable progress toward elucidating post-receptor mechanisms in the biochemical pathways of hormone action. At present, there are a limited number of examples of mutations in genes encoding proteins that function in this part of the pathway, but it seems likely that additional examples will be discovered in the future. It is likely that these insights into biochemical mechanisms of disease will ultimately lead to an improvement in our ability to treat human disease.

Genetic Diseases, Inborn↗
PubMed1994

Advances in kinetic analysis of insulin-stimulated GLUT-4 translocation in adipose cells.

GLUT-4 is the major insulin-sensitive glucose transporter in muscle and adipose tissue. Regulation of GLUT-4 is an important component of whole body glucose homeostasis. Abnormalities in the regulation of insulin-stimulated reversible translocation of glucose transporters have been observed in various pathological states, including diabetes. Recently, the development of specific photolabels for glucose transporters and the availability of antibodies against the various transporter isoforms have presented the opportunity for detailed kinetic analysis of GLUT-4 regulation. A kinetic analysis of some of the most recent data is presented to demonstrate how this approach can advance the understanding of GLUT-4 regulation. Some areas in which the currently available data limit the ability to resolve certain mechanistic questions are noted. Using a two-compartment model, we show that the mechanism of insulin-stimulated GLUT-4 translocation is likely to involve a large increase in the exocytosis rate of GLUT-4 with a minimal decrease in the endocytosis rate. Mathematical models based on these kinetic analyses are helpful for testing hypotheses and designing experiments to elucidate the molecular and cellular mechanisms of GLUT-4 regulation under normal and pathological conditions. This type of approach may be useful for evaluating the contribution of GLUT-4 regulation to the pathogenesis of diabetes.

Adipocytes↗
PubMed1994

Non-insulin-mediated glucose disappearance in subjects with IDDM. Discordance between experimental results and minimal model analysis.

Both insulin and glucose contribute to the regulation of glucose metabolism in vivo. We directly measured the ability of glucose per se to promote glucose disposal in subjects with insulin-dependent diabetes mellitus (IDDM). We compared our results with predictions of the minimal model of glucose metabolism. To identify minimal model parameters, a frequently sampled intravenous glucose tolerance test (FSIVGTT) was administered to each subject while they were connected to a Biostator (a device that monitors blood glucose and gives insulin to mimic normal insulin secretion). Data from this test reflected normal glucose tolerance and were in excellent agreement with minimal model predictions. The FSIVGTT was then repeated without the Biostator in the same diabetic subjects in order to directly measure the effect of glucose per se to promote glucose disposal in the absence of an incremental insulin effect (a basal insulin drip was maintained). To compare these results with minimal model predictions, the equations describing glucose disappearance in the absence of an incremental insulin effect were solved using parameters identified from the Biostator experiment. The glucose disappearance measured in the absence of an incremental insulin response was much slower than the minimal model predictions. Thus, the minimal model appears to overestimate the effect of glucose per se on glucose uptake and underestimate the contribution of incremental insulin.

Adult↗
PubMed1994

Direct comparison of standard and insulin modified protocols for minimal model estimation of insulin sensitivity in normal subjects.

Estimates of in vivo insulin sensitivity (S1) can be derived from minimal model analysis of a frequently sampled intravenous glucose tolerance test (FSIVGTT). Modification of the FSIVGTT by administration of tolbutamide improves the correlation between S1 and more traditional glucose clamp measurements. Recently, a modified FSIVGTT using insulin infusion (instead of tolbutamide) has been described. This modification may be useful for studying subjects with poor insulin secretion. We directly compare the standard FSIVGTT with the insulin modified FSIVGTT in ten normal subjects (age 29 +/- 2 yr, BMI 22.5 +/- 1.2 kg/m2). Each subject received both tests. The order of the studies was randomized and the interval between paired studies was approximately one week. After an overnight fast, glucose (0.3 g/kg) was infused from time 0 to 2 min. For the modified FSIVGTT, insulin (4 mUkg-1.min-1) was infused from time 20 to 25 min. None of the subjects became hypoglycaemic after insulin infusion. Paired t-test analysis did not reveal significant differences between the two protocols for any of the minimal model parameters. The standard FSIVGTT gave estimates for S1 of 5.63 +/- 1.32 x 10(-4) min-1 per microU/ml while the insulin modified FSIVGTT gave estimates for S1 of 5.11 +/- 0.83 x 10(-4) min-1 per microU/ml. However, the fractional standard deviation for S1 was significantly smaller with the insulin modified protocol (3.6 +/- 1.0 vs. 22.2 +/- 9.0, p < 0.037). Thus, under our experimental conditions, insulin infusion during FSIVGTT appears to improve the precision of minimal model estimation of S1 in normal subjects.

Adult↗
PubMed1993

Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action.

The subcellular trafficking of tracer-tagged GLUT4 between the plasma membranes and low-density microsomes of rat adipose cells has been studied. Cell-surface GLUT4 have been initially tracer-tagged in the insulin-stimulated state with the [3H]bismanose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine. The half-time for internalization of tracer-tagged GLUT4 when insulin is removed by collagenase treatment is similar to that observed for the decrease in immunodetectable GLUT4 in the plasma membranes and the decrease in glucose transport activity in the intact cells. In contrast, internalization of tracer-tagged GLUT4 also occurs when cells are maintained in the continuous presence of insulin even though the plasma membrane level of immunodetectable GLUT4 and glucose transport activity in the intact cells are unaltered. These data show, for the first time, that insulin has little, if any, effect on the rate constant for GLUT4 endocytosis, but instead, primarily increases the rate constant for exocytosis. Tracer-tagged GLUT4 that is returned to the low-density microsomes can be restimulated with fresh insulin to recycle to the plasma membranes and to a steady-state distribution level that is the same as that observed in cells that are maintained in the continuous presence of insulin. These data suggest that the cells' entire complement of GLUT4 is involved in the recycling process. Following insulin stimulation of adipose cells initially in the basal state, the increase in immunodetectable GLUT4 in the plasma membranes precedes the increase in accessibility of GLUT4 to exofacial 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine photolabeling, and this in turn precedes the increase in cellular glucose transport activity. Such time course data suggest that there may be plasma membrane intermediate states in the GLUT4 trafficking pathway. The kinetic properties of GLUT4 translocation and its recycling have been interpreted in terms of a subcellular trafficking model that identifies exocytosis, possibly involving-hypothetical "docking" and "fusion" steps, as the critical site of hormone action.

3-O-Methylglucose↗
PubMed1993

Transfection of DNA into isolated rat adipose cells by electroporation: evaluation of promoter activity in transfected adipose cells which are highly responsive to insulin after one day in culture.

Isolated adipose cells are among the most insulin responsive cells with respect to glucose transport and metabolism. However, molecular biological techniques such as transfection of DNA have heretofore not been applied successfully in these cells in primary culture. We report a method for transfection of DNA into rat adipose cells by electroporation. Six shocks at 800 V and 25 microF in a 0.4 cm gap cuvette results in efficient transfection. We compared the ability of five promoters to drive expression of a luciferase reporter gene in transfected adipose cells. After one day in culture, promoter activity ranged from no expression to a very high level of expression. These transfected, cultured cells also displayed a 10-fold increase in 3-O-methylglucose transport with maximal insulin stimulation. The ability to transfect DNA into adipose cells which remain insulin responsive after one day in primary culture may be helpful for understanding adipose cell-specific gene regulation and elucidating the molecular mechanisms of insulin action.

3-O-Methylglucose↗
PubMed1993

Substitution of glutamic acid for alanine 1135 in the putative "catalytic loop" of the tyrosine kinase domain of the human insulin receptor. A mutation that impairs proteolytic processing into subunits and inhibits receptor tyrosine kinase activity.

The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase which is stimulated by insulin binding to the extracellular domain of the receptor. We have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Glu for Ala1135 in the putative "catalytic loop" of the tyrosine kinase domain of the receptor. In this investigation, the Glu1135 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. Like previously described mutations in the tyrosine kinase domain, the Glu1135 mutation impairs receptor tyrosine kinase activity and inhibits the ability of insulin to stimulate thymidine incorporation and receptor endocytosis. These data support the hypothesis that the receptor tyrosine kinase activity plays a necessary role in the ability of the receptor to mediate insulin action in vitro and in vivo. However, unlike previously described mutations in the intracellular domain of the receptor, the Glu1135 mutation impairs proteolytic cleavage of the proreceptor into separate subunits and impairs the transport of the receptor to the cell surface. These latter defects provide an explanation for the decrease in the number of receptors on the cell surface observed in the patient's circulating monocytes despite the fact that the mutant receptor is resistant to endocytosis and insulin-induced down-regulation.

3T3 Cells↗
PubMed1992

Postbinding characterization of five naturally occurring mutations in the human insulin receptor gene: impaired insulin-stimulated c-jun expression and thymidine incorporation despite normal receptor autophosphorylation.

Some patients with extreme insulin resistance have mutations in their insulin receptor gene. We previously identified five such mutations located in the extracellular domain of the insulin receptor (Asn-->Lys15, His-->Arg209, Phe-->Val382, Lys-->Glu460, and Asn-->Ser462) and studied the effects of these mutations upon posttranslational processing, insulin binding, and tyrosine autophosphorylation. We now characterize the ability of these mutant receptors to mediate biological actions of insulin in transfected NIH-3T3 fibroblasts. All cell lines expressing mutant receptors showed marked impairment in insulin-stimulated c-jun expression and thymidine incorporation when compared with cells expressing wild-type human insulin receptors. The most severe impairment was seen in cells expressing the Val382 mutant (a mutation which causes an intrinsic defect in receptor autophosphorylation). These cells had insulin responses similar to the untransfected cells (used as a negative control). In contrast, cells expressing the Lys15 mutant have the ability to achieve a normal level of maximal autophosphorylation but require an abnormally high concentration of insulin to do so (as the result of decreased insulin binding affinity). These cells show a higher basal rate and much lower insulin stimulation of both c-jun expression and thymidine incorporation when compared with the cells expressing the wild-type human insulin receptors. This pattern is also seen in the cells expressing the other mutants with normal autophosphorylation (Arg209, Glu460, and Ser462). Although the most severe defects in insulin action are seen with the mutation which has an intrinsic defect in receptor autophosphorylation, the ability to undergo normal autophosphorylation does not seem to preclude mutations from impairing the ability of receptors to mediate some of the actions of insulin.

Cell Line↗
PubMed1992

Substitution of isoleucine for methionine at position 1153 in the beta-subunit of the human insulin receptor. A mutation that impairs receptor tyrosine kinase activity, receptor endocytosis, and insulin action.

The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase. The receptor tyrosine kinase is stimulated by insulin binding to the extracellular domain of the receptor. Previously, we have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Ile for Met1153 in the tyrosine kinase domain of the receptor near the cluster of the three major autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163). In this investigation, the Ile1153 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs receptor tyrosine kinase activity and also inhibits the ability of insulin to stimulate 2-deoxyglucose uptake and thymidine incorporation. These data support the hypothesis that the receptor tyrosine activity plays a necessary role in the ability of the receptor to mediate insulin action in vivo. Furthermore, expression of the Ile1153 mutant receptor exerted a dominant negative effect to inhibit the ability of endogenous murine receptors for insulin and insulin-like growth factor I to mediate their actions upon the cell. This observation is consistent with previous suggestions that mutant receptors dimerize with wild type receptors, thereby creating hybrid molecules which lack biological activity. The dominant negative effect of the mutant receptor may explain the dominant mode of inheritance of insulin resistance caused by the Ile1153 mutation. Finally, the mutation inhibits the ability of insulin to stimulate receptor endocytosis. This may explain the normal number of insulin receptors on the surface of the patient's cells in vivo. Despite the presence of markedly elevated levels of insulin in the patient's plasma, the receptors were resistant to down-regulation.

3T3 Cells↗