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Biomedical subjects

I D Goldfine

Publications and source records attributed to I D Goldfine.

At least 19 recordsLinked to original sources
PubMed2000

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.

Cell Line↗
PubMed1999

Insulin receptor activation by IGF-II in breast cancers: evidence for a new autocrine/paracrine mechanism.

IGF-II, produced by breast cancer epithelial and stromal cells, enhances tumor growth by activating the IGF-I receptor (IGF-I-R) via autocrine and paracrine mechanisms. Previously we found that the insulin receptor (IR), which is related to the IGF-I-R, is overexpressed in breast cancer cells. Herein, we find that, in breast cancer the IR is activated by IGF-II. In eight human breast cancer cell lines studied there was high affinity IGF-II binding to the IR, with subsequent IR activation. In these lines, IGF-II had a potency up to 63% that of insulin. In contrast, in non malignant human breast cells, IGF-II was less than 1% potent as insulin. Via activation of the IR tyrosine kinase IGF-II stimulated breast cancer cell growth. Moreover, IGF-II also activated the IR in breast cancer tissue specimens; IGF-II was 10-100% as potent as insulin. The IR occurs in two isoforms generated by alternative splicing of exon 11; these isoforms are IR-A (Ex11-) and IR-B (Ex11+). IR-A was predominantly expressed in breast cancer cells and specimens and the potency of IGF-II was correlated to the expression of this isoform (P<0.0001). These data indicate, therefore, that the IR-A, which binds IGF-II with high affinity, is predominantly expressed in breast cancer cells and represents a new autocrine/paracrine loop involved in tumor biology.

Binding, Competitive↗
PubMed1999

Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells.

Insulin-like growth factor II (IGF-II) is a peptide growth factor that is homologous to both insulin-like growth factor I (IGF-I) and insulin and plays an important role in embryonic development and carcinogenesis. IGF-II is believed to mediate its cellular signaling via the transmembrane tyrosine kinase type 1 insulin-like growth factor receptor (IGF-I-R), which is also the receptor for IGF-I. Earlier studies with both cultured cells and transgenic mice, however, have suggested that in the embryo the insulin receptor (IR) may also be a receptor for IGF-II. In most cells and tissues, IR binds IGF-II with relatively low affinity. The IR is expressed in two isoforms (IR-A and IR-B) differing by 12 amino acids due to the alternative splicing of exon 11. In the present study we found that IR-A but not IR-B bound IGF-II with an affinity close to that of insulin. Moreover, IGF-II bound to IR-A with an affinity equal to that of IGF-II binding to the IGF-I-R. Activation of IR-A by insulin led primarily to metabolic effects, whereas activation of IR-A by IGF-II led primarily to mitogenic effects. These differences in the biological effects of IR-A when activated by either IGF-II or insulin were associated with differential recruitment and activation of intracellular substrates. IR-A was preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney and had a relatively increased proportion of isoform A. IR-A expression was also increased in several tumors including those of the breast and colon. These data indicate, therefore, that there are two receptors for IGF-II, both IGF-I-R and IR-A. Further, they suggest that interaction of IGF-II with IR-A may play a role both in fetal growth and cancer biology.

3T3 Cells↗
PubMed1999

Insulin receptor autophosphorylation in cultured myoblasts correlates to glucose disposal in Pima Indians.

In a previous study [Youngren, J. F., I. D. Goldfire, and R. E. Pratley. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E276-E283, 1997] of skeletal muscle biopsies from insulin-resistant, nondiabetic Pima Indians, we demonstrated that diminished insulin receptor (IR) autophosphorylation correlated with in vivo insulin resistance. In the present study, to determine whether decreased IR function is a primary trait of muscle, and not secondary to an altered in vivo environment, we cultured myoblasts from 17 nondiabetic Pima Indians in whom insulin-stimulated glucose disposal (M) was measured during hyperinsulinemic-euglycemic glucose clamps. Myoblast IR autophosphorylation was determined by a highly sensitive ELISA. IR autophosphorylation directly correlated with M (r = 0.56, P = 0.02) and inversely correlated with the fasting plasma insulin (r = -0.58, P < 0.05). The relationship between M and IR autophosphorylation remained significant after M was adjusted for the effects of percent body fat (partial r = 0.53, P < 0.04). The relationship between insulin resistance and the capacity for myoblast IR autophosphorylation in nondiabetic Pima Indians suggests that variations in IR-signaling capacity may be intrinsic characteristics of muscle that contribute to the genetic component determining insulin action in this population.

Adult↗
PubMed1999

A polymorphism (K121Q) of the human glycoprotein PC-1 gene coding region is strongly associated with insulin resistance.

The genes responsible for insulin resistance are poorly defined. Plasma cell differentiation antigen (PC-1) glycoprotein inhibits insulin receptor signaling and is associated with insulin resistance. We describe here a novel polymorphism in exon 4 of the PC-1 gene (K121Q) and demonstrate that it is strongly associated with insulin resistance in 121 healthy nonobese (BMI <30 kg/m2) nondiabetic (by oral glucose tolerance test [OGTT]) Caucasians from Sicily. Compared with 80 KK subjects, Q allele carriers (n = 41, 39 KQ and 2 QQ) showed higher glucose and insulin levels during OGTT (P < 0.001 by two-way analysis of variance) and insulin resistance by euglycemic clamp (M value = 5.25 +/- 1.38 [n = 24] vs. 6.30 +/- 1.39 mg x kg(-1) x min(-1) [n = 49], P = 0.005). Q carriers had higher risk of being hyperinsulinemic and insulin resistant (odds ratio [CI]: 2.99 [1.28-7.0], P < 0.001). Insulin receptor autophosphorylation was reduced (P < 0.01) in cultured skin fibroblasts from KQ versus KK subjects. Skeletal muscle PC-1 content was not different in 11 KQ versus 32 KK subjects (33 +/- 16.1 vs. 17.5 +/- 15 ng/mg protein, P = 0.3). These results suggest a cause-effect relationship between the Q carrying genotype and the insulin resistance phenotype, and raise the possibility that PC-1 genotyping could identify individuals who are at risk of developing insulin resistance, a condition that predisposes to type 2 diabetes and coronary artery disease.

Adult↗
PubMed1999

Insulin and insulin-like growth factor-I (IGF-I) receptor overexpression in breast cancers leads to insulin/IGF-I hybrid receptor overexpression: evidence for a second mechanism of IGF-I signaling.

The insulin receptor (IR) form hybrids with the closely related insulin-like growth factor-I (IGF-I) receptor (IGF-I-R). Because most human breast carcinomas overexpress both the IR and the IGF-I-R, we evaluated whether the insulin/IGF-I hybrid receptor (Hybrid-R) is also overexpressed in these tumors and what role it plays in breast cancer biology. Using specific ELISAs and Western blots, we measured Hybrid-R content and function in 8 human cultured breast cancer cell lines and 39 human breast cancer specimens. Hybrid-R content and function were also compared to the content and function of the IR and the IGF-I-R. Hybrid-R content exceeded the IGF-I-R content in >75% of breast cancer specimens and was directly related to the molar ratio of both the IR and IGF-I-R content, suggesting that Hybrid-R formation occurred by random assembly of IR and IGF-I-R half-receptors. Hybrid-Rs became tyrosine autophosphorylated when breast cancer cells were exposed to IGF-I but not when they were exposed to insulin. In cells with an elevated Hybrid-R content, Hybrid-R autophosphorylation in response to IGF-I exceeded IGF-I-R autophosphorylation, suggesting that most of the IGF-I effect occurred via the Hybrid-R. Furthermore, Hybrid-Rs mediated growth in response to IGF-I, as indicated by experiments with blocking antibodies to the IGF-I-R. These data indicated therefore that: (a) Hybrid-Rs are present and play a major role in mediating the IGF-I signal in breast cancer; (b) their expression is directly related to IR overexpression; and (c) potential therapies designed to block IGF-I actions in breast cancer must take into account the role of these Hybrid-Rs.

Antibodies, Monoclonal↗
PubMed1998

Insulin-stimulated cell growth in insulin receptor substrate-1-deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase-independent pathway.

In many human breast cancers and cultured cell lines, insulin receptor expression is elevated, and insulin, via its own insulin receptor, can stimulate cell growth. It has recently been demonstrated that the enzyme phosphatidylinositol-3-kinase (PI3-K) mediates various aspects of insulin receptor signaling including cell growth. In order to understand the mechanisms for insulin-stimulated cell growth in human breast cancer, we measured insulin-stimulable PI3-K activity in a non-transformed breast epithelial cell line, MCF-10A, and in two malignantly transformed cell lines, ZR-75-1 and MDA-MB157. All three cell lines express comparable amounts of insulin receptors whose tyrosine autophosphorylation is increased by insulin, and in these cell lines insulin stimulates growth. In MDA-MB157 and MCF-10A cells, insulin stimulated PI3-K activity three- to fourfold. In ZR-75-1 cells, however, insulin did not stimulate PI3-K activity. In ZR-75-1 cells PI3-K protein was present, and its activity was stimulated by epidermal growth factor, suggesting that there might be a defect in insulin receptor signaling upstream of PI3-K and downstream of the insulin receptor. Next, we studied insulin receptor substrate-1 (IRS-1), a major endogenous substrate for the insulin receptor which, when tyrosine is phosphorylated by the insulin receptor, interacts with and activates PI3-K. In ZR-75-1 cells, there were reduced levels of protein for IRS-1. In these cells, both Shc tyrosine phosphorylation and mitogen-activated protein kinase (MAP-K) activity were increased by the insulin receptor (indicating that the p21ras pathway may account for insulin-stimulated cell growth in ZR-75-1 cells). The PI3-K inhibitor LY294002 (50 microM) reduced insulin-stimulated growth in MCF-10A and MDA-MB157 cell lines, whereas it did not modify insulin effect on ZR-75-1 cell growth. The MAP-K/Erk (MEK) inhibitor PD98059 (50 microM) consistently reduced insulin-dependent growth in all three cell lines. Taken together, these data suggest that in breast cancer cells insulin may stimulate cell growth via PI3-K-dependent or-independent pathways.

Breast Neoplasms↗
PubMed1998

Contributions of the American Journal of Physiology to the discovery of insulin.

Since its inception in 1898 the American Journal of Physiology has been a leader in diabetes research and has published many key articles on the subject. The Journal first published studies of phlorhizin-induced diabetes in 1898, and after many other contributions went on to publish the first reports of Banting, Best, Macleod, and Collip in 1922 concerning the isolation and purification of insulin (5-8, 13). This review highlights some of these key contributions of the Journal.

Animals↗
PubMed1998

Elevated PC-1 content in cultured skin fibroblasts correlates with decreased in vivo and in vitro insulin action in nondiabetic subjects: evidence that PC-1 may be an intrinsic factor in impaired insulin receptor signaling.

Membrane glycoprotein PC-1 inhibits insulin receptor (IR) tyrosine kinase activity and subsequent cellular signaling. PC-1 content is elevated in muscle and adipose tissue from insulin-resistant subjects, and its elevation correlates with in vivo insulin resistance. To determine whether elevated PC-1 content is a primary cause of insulin resistance, we have now measured PC-1 content in cultured skin fibroblasts from nonobese nondiabetic insulin-resistant subjects and found that 1) PC-1 content was significantly higher in these cells when compared with cells from insulin-sensitive subjects (6.7 +/- 0.9 vs. 3.1 +/- 0.6 ng/0.1 mg protein, mean +/- SE, P < 0.01); 2) PC-1 content in fibroblasts was highly correlated with PC-1 content in muscle tissue (r = 0.95, P = 0.01); 3) PC-1 content in fibroblasts negatively correlated with both decreased in vivo insulin sensitivity and decreased in vitro IR autophosphorylation; and 4) in cells from insulin-resistant subjects, insulin stimulation of glycogen synthetase was decreased. These studies indicate, therefore, that the elevation of PC-1 content may be a primary factor in the cause of insulin resistance.

Adult↗
PubMed1998

Membrane glycoprotein PC-1 and insulin resistance.

Peripheral resistance to insulin is a major component of non-insulin dependent diabetes mellitus. Defects in insulin receptor tyrosine kinase activity have been demonstrated in several tissues from insulin resistant subjects, but mutations in the insulin receptor gene occur in only a small fraction of cases. Therefore, other molecules that are capable of modulating the function of the insulin receptor are likely candidates in the search for the cellular mechanisms of insulin resistance. We have isolated an inhibitor of insulin receptor tyrosine kinase activity from cultured fibroblasts of an insulin resistant NIDDM patient and identified it as membrane glycoprotein PC-1. Subsequently we have demonstrated that expression of PC-1 is elevated in fibroblasts from other insulin resistant subjects, both with and without NIDDM. Studies in muscle, the primary site for insulin-mediated glucose disposal, have shown that the levels of PC-1 in this tissue are inversely correlated to insulin action both in vivo and in vitro. Transfection of PC-1 into cultured cells has confirmed that overexpression of PC-1 can produce impairments in insulin receptor tyrosine kinase activity and the subsequent cellular responses to insulin. Preliminary data suggests a direct interaction between PC-1 and the insulin receptor. However, the mechanisms whereby PC-1 inhibits insulin receptor signaling remain to be determined.

Animals↗
PubMed1997

ASPB10 insulin induction of increased mitogenic responses and phenotypic changes in human breast epithelial cells: evidence for enhanced interactions with the insulin-like growth factor-I receptor.

The human insulin analogue ASPB10 has been reported to have increased affinity for the insulin receptor and to cause breast cancer in female rats. In the study reported here, we investigated whether ASPB10 has an increased mitogenic potency and induces a transformed phenotype in cultured human breast cells. In both MCF-10 cells (a non-malignant human breast line) and MCF-7 cells (a human breast cancer cell line), ASPB10 was approximately twofold more potent than insulin in competing for 125I-insulin binding but sevenfold to tenfold more potent than insulin in competing for 125I-insulin-like growth factor (IGF)-I binding. In addition, ASPB10 was twofold more potent than insulin in stimulating insulin receptor autophosphorylation but significantly more potent in stimulating IGF-I receptor autophosphorylation in both cell lines. Moreover, ASPB10 was approximately sevenfold more potent than insulin in stimulating the growth of MCF-10 and MCF-7 cells. This increased mitogenic effect of ASPB10 was significantly inhibited (but not abolished) when cells were cultured in the presence of alpha-IR3, a monoclonal antibody to the IGF-I receptor. ASPB10, but not insulin, caused phenotypic changes (focus formation) in MCF-10 cells. Neither agent caused colony formation in soft agar in MCF-10 cells, but ASPB10 was more potent than insulin in stimulating colony formation in MCF-7 cells. These observations indicate that in human breast cells, ASPB10 has enhanced mitogenic effects and induces phenotypic changes as a consequence of its activation of both insulin and IGF-I receptors.

Binding, Competitive↗
PubMed1997

Sporadic amplification of the insulin receptor gene in human breast cancer.

Insulin receptor (IR) content is increased in most human breast carcinomas when compared to normal breast tissue. In the present study we investigated IR gene copy number by using both conventional DNA analysis (slot blot) and fluorescence in situ hybridization (FISH). Cultured human breast cell lines and primary breast carcinoma specimens were analyzed. In 6 breast cell lines in culture both techniques gave similar results: the relative IR copy number determined by FISH strongly correlated with slot blot results (r = 0.831), even if probes for different reference loci were used in the 2 methods. We find that in human breast cancer IR gene amplification is a sporadic event. It occurred in 1/5 cultured breast cancer cell lines (MDA-MB 231) and in 8/93 (8.6%) breast cancer specimens. In contrast an increased copy number of the entire chromosome 19 (which contains IR gene) was frequently observed in both breast cancer cell lines (100%) and breast cancer specimens (45%). When present, IR gene amplification always occurred at low level. These data indicate that IR gene amplification is an uncommon event in human breast carcinomas and that mechanisms other than gene amplification are responsible for IR protein overexpression in most human breast cancers.

Blotting, Southern↗
PubMed1997

Increased adipose tissue PC-1 protein content, but not tumour necrosis factor-alpha gene expression, is associated with a reduction of both whole body insulin sensitivity and insulin receptor tyrosine-kinase activity.

In the present study we measured PC-1 content, tumour necrosis factor (TNF)-alpha gene expression, and insulin stimulation of insulin receptor tyrosine-kinase activity in adipose tissue from non-obese, non-diabetic subjects. These parameters were correlated with in vivo insulin action as measured by the intravenous insulin tolerance test (Kitt values). PC-1 content was negatively correlated with Kitt values (r = -0.5, p = 0.04) and positively with plasma insulin levels both fasting (r = 0.58, p = 0.009) and after 120 min during oral glucose tolerance test (OGTT) (r = 0.67, p = 0.002). Moreover, adipose tissue PC-1 content was higher in relatively insulin-resistant subjects (Kitt values lower than 6) than in relatively insulin-sensitive subjects (Kitt values higher than 6) (525 +/- 49 ng/mg protein vs 336 +/- 45, respectively, p = 0.012). Adipose tissue insulin receptor tyrosine-kinase activity in response to insulin was significantly lower at all insulin concentrations tested (p = 0.017, by two-way analysis of variance test) in insulin-resistant than in insulin-sensitive subjects (Kitt values lower or higher than 6, respectively). In contrast to PC-1, no significant correlation was observed between adipose tissue TNF-alpha mRNA content and Kitt values, and plasma insulin levels, both fasting and at after 120 min during OGTT. Also, no difference was observed in TNF-alpha mRNA content between subjects with Kitt values higher or lower than 6. These studies in adipose tissue, together with our previous studies in skeletal muscle raise the possibility that PC-1, by regulating insulin receptor function, may play a role in the degree of insulin sensitivity in non-obese, non-diabetic subjects.

Adipose Tissue↗
PubMed1997

The insulin receptor content is increased in breast cancers initiated by three different oncogenes in transgenic mice.

The Insulin Receptor (IR) is a potential oncogene for mammary epithelial cells since its content is increased in most human breast cancer specimens, and both ligand-dependent malignant transformation and ligand-dependent enhanced growth occurs in cultured breast cells overexpressing the IR. To better understand whether the IR plays a role in mammary carcinogenesis which is independent of other initiation factors, we measured IR content in transgenic mouse models of breast cancer induced by 3 known oncogenes (Wnt-1, Neu, and Ret). Insulin receptor content was measured by a specific radioimmunoassay. In normal mammary gland tissues IR content was 14.6 +/- 1.4 ng/mg of protein (mean +/- SEM, n = 6). In the 3 cancers IR content was elevated (Neu = 36.1 +/- 4.6, n = 8, p < 0.002; Wnt-1 = 38.3 +/- 2.6, n = 13, p < 0.001; and Ret = 53.6 +/- 7.1, n = 7, p < 0.001). These data indicate that IR overexpression, in addition to being a potential oncogene, is increased in mouse tumors initiated by other oncogenes, and therefore may also play a supportive role in the growth of breast cancers.

Animals↗
PubMed1997

The endocrine secretion of human insulin and growth hormone by exocrine glands of the gastrointestinal tract.

The exocrine pancreas, liver, and submandibular glands of the rat were used to express and secrete two exogenous, human protein hormones (growth hormone and insulin) into blood at physiological concentrations. Transfection, expression, and secretion were achieved by the in vivo retrograde injection of plasmid DNA into the secretory ducts of these glands. Pancreatic acinar cells secreted physiological concentrations of growth hormone into the circulation, and its secretion was enhanced by cholinergic stimulation. A human insulin gene was engineered to allow normal processing of insulin in non-beta cells. With this gene, the secretion of human insulin by the exocrine pancreas normalized elevated blood glucose levels in diabetic rats. These in vivo observations demonstrate the utility of retrograde ductal administration of naked DNA into exocrine organs as a novel method for the regulated systemic delivery of protein-based pharmaceuticals.

Animals↗
PubMed1997

Decreased muscle insulin receptor kinase correlates with insulin resistance in normoglycemic Pima Indians.

Defects in insulin receptor tyrosine kinase activity are present in insulin-resistant non-insulin-dependent diabetes mellitus patients and certain nondiabetic individuals, both lean and obese. However, the relationship between insulin receptor function, insulin action, and obesity is unclear. To address this issue, we have employed a new and highly sensitive enzyme-linked immunosorbent assay to measure in vitro insulin-stimulated autophosphorylation of immunocaptured muscle insulin receptors in a group of 25 normoglycemic Pima Indians. Insulin action, determined during two-step euglycemic insulin clamps, varied widely in these subjects. Maximal in vitro insulin stimulation of insulin receptor autophosphorylation strongly correlated with both low (Mlow)- and high (Mhigh)-dose insulin-stimulated glucose disposal (r = 0.62 and 0.51, P < 0.002 and 0.011, respectively). Insulin receptor autophosphorylation was inversely related to percent body fat (r = -0.52, P < 0.009). After control for percent body fat, receptor autophosphorylation remained correlated with Mlow (partial r = 0.49, P < 0.025). These data therefore suggest that defects in insulin receptor function are major contributors to insulin resistance in both lean and obese normoglycemic Pima Indians.

Adult↗
PubMed1997

Insulin receptor expression and clinical outcome in node-negative breast cancer.

The insulin receptor (IR), a ligand-activated tyrosine kinase, is present in breast cancers, but its relationship to patient survival is unknown. The IR was measured in 584 tumor specimens from patients with node-negative breast carcinoma by frozen-section immunohistochemistry and light microscopy. The immunostaining signal was quantitated in relation to both the staining intensity and the proportion of positive malignant epithelial cells. Analyses indicated that patients with tumors with undetectable IR content in malignant epithelial cells (260 cases) had a relatively lower predicted 5-year disease-free survival (DFS) (69% +/- 3%) than did patients with tumors with detectable IR content (324 cases; DFS 76% +/- 3%, p = .032). The significance of IR content in these breast malignant epithelial cells was then analyzed along with patient age, tumor size, progesterone and estrogen receptor status, p53 accumulation, and S-phase. Multivariate analysis of these data revealed that after adjustment for these other variables, IR content was the strongest independent predictive factor for DFS (relative risk = 1.73, p = .005). Interestingly, in a small subset of patients with very high IR content (n = 62), DFS was decreased. These data indicate that IR content in node-negative breast cancers is a significant major predictor of reduced DFS. Moreover, they raise the possibility that the measurement of IR content might provide important information concerning breast cancer biology.

Adult↗