Insulin, insulin receptors, and cancer.

Insulin is a major regulator of cell metabolism but, in addition, is also a growth factor. Insulin effects in target cells are mediated by the insulin receptor (IR), a transmembrane protein with enzymatic (tyrosine kinase) activity. The insulin receptor, however, is represented by a heterogeneous family of proteins, including two different IR isoforms and also hybrid receptors resulting from the IR hemireceptor combination with a hemireceptor of the cognate IGF-1 receptor. These different receptors may bind insulin and its analogs with different affinity and produce different biologic effects. Since many years, it is known that many cancer cells require insulin for optimal in vitro growth. Recent data indicate that: (1) insulin stimulates growth mainly via its own receptor and not the IGF-1 receptor; (2) in many cancer cells, the IR is overexpressed and the A isoform, which has a predominant mitogenic effect, is more represented than the B isoform. These characteristics provide a selective growth advantage to malignant cells when exposed to insulin. For this reason, all conditions of hyperinsulinemia, both endogenous (prediabetes, metabolic syndrome, obesity, type 2 diabetes before pancreas exhaustion and polycystic ovary syndrome) and exogenous (type 1 diabetes) will increase the risk of cancer. Cancer-related mortality is also increased in patients exposed to hyperinsulinemia but other factors, related to the different diseases, may also contribute. The complexity of the diseases associated with hyperinsulinemia and their therapies does not allow a precise evaluation of the cancer-promoting effect of hyperinsulinemia, but its detrimental effect on cancer incidence and mortality is well documented.

XMetA, an allosteric monoclonal antibody to the insulin receptor, improves glycaemic control in mice with diet-induced obesity.

XMetA, a high-affinity, fully human monoclonal antibody, allosterically binds to and activates the insulin receptor (INSR). Previously, we found that XMetA normalized fasting glucose and glucose tolerance in insulinopenic mice. To determine whether XMetA is also beneficial for reducing hyperglycaemia due to the insulin resistance of obesity, we have now evaluated XMetA in hyperinsulinemic mice with diet-induced obesity. XMetA treatment of these mice normalized fasting glucose for 4 weeks without contributing to weight gain. XMetA also corrected glucose tolerance and improved non-high density lipoprotein cholesterol. These studies indicate, therefore, that monoclonal antibodies that allosterically activate the INSR, such as XMetA, have the potential to be novel agents for the treatment of hyperglycaemia in conditions associated with the insulin resistance of obesity.

Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells.

Previous studies have suggested a role for glucocorticoids in the differentiation of the acinar pancreas. We have now used the rat tumor cell line AR42J, derived from the acinar pancreas, to directly study this effect of glucocorticoids in vitro. The steroid hormones dexamethasone, corticosterone, aldosterone, and progesterone, but not estrogen, increased both the amylase content and the number of secretory granules of these cells. The potencies of the steroids were directly related to their effectiveness as glucocorticoids; dexamethasone was the most potent hormone and gave maximal effects at 100 nM. Morphometric analyses revealed that dexamethasone increased the volume density of granules 5.5-fold from 0.20 +/- 0.08 to 1.10 +/- 0.20% (n = 4) of the cytoplasmic volume. Dexamethasone treatment also increased the volume density of rough endoplasmic reticulum 2.4-fold from 1.20 +/- 0.09 to 2.86 +/- 0.30% (n = 5) of the cytoplasmic volume. After 48 h of dexamethasone treatment the cellular content of amylase increase eightfold from 2.8 +/- 0.4 to 22.6 +/- 3.8 U/mg protein (n = 6). This effect of dexamethasone was discernible after 12 h of incubation and approached maximal stimulation after 72 h of incubation. The increases in cellular amylase content were due to increased amylase synthesis as shown by specific immunoprecipitation of [35S]methionine-labeled proteins. Moreover, in vitro translation of cellular mRNA indicated that dexamethasone treatment increased amylase mRNA. Dexamethasone treatment also led to increased secretion of amylase in response to the secretagogue cholecystokinin. These data indicate, therefore, that glucocorticoids induce a more highly differentiated phenotype in AR42J pancreatic cells, and they suggest that glucocorticoids act via the enhanced transcription of specific mRNAs for acinar cell proteins.

Insulin receptor antiserum and plant lectins mimic the direct effects of insulin on nuclear envelope phosphorylation.

Insulin directly inhibits protein phosphorylation in isolated rat liver nuclear envelopes. In the present studies, an antiserum to insulin receptor as well as the plant lectins concanavalin A and phytohemagglutinin mimicked insulin action in isolated nuclear envelopes. These studies suggest that insulin and agents that mimic it may directly regulate nuclear functions.

Cholecystokinin receptors in the brain: characterization and distribution.

Specific cholecystokinin binding sites in particulate fractions of rat brain were measured with iodine 125-labeled Bolton-Hunter cholecystokinin, a cholecystokinin analog that has full biological activity. Binding was detected in brain regions known to contain immunoreactive cholecystokinin. Binding was saturable, reversible, of high affinity (dissociation constant, 1.7 x 10(-9) M), and was inhibited by cholecystokinin analogs but not by unrelated hormones.

Insulin stimulation of nucleoside triphosphatase activity in isolated nuclear envelopes.

The activity of nucleoside triphosphatase, an enzyme that regulates nuclear messenger RNA transport, was measured in highly purified nuclear envelopes isolated from rat liver. Addition of picomolar concentrations of insulin to freshly prepared nuclear envelopes directly increased the enzyme activity. The major effect of insulin on this enzyme was to increase the maximum velocity of its activity; no significant effects were seen on the affinity constant. These studies raise the possibility, therefore, that the nuclear envelope is a site where insulin regulates nuclear functions.

Entry of insulin into human cultured lymphocytes: electron microscope autoradiographic analysis.

Electron microscope autoradiographs were prepared of IM-9 human cultured lymphocytes incubated with iodine-125-labeled insulin. With the use of [125I]insulin and Ilford L-4 emulsion, the technique had a resolution half-distance of approximately 0.085 micrometer. Autoradiographs revealed a time-dependent entry of insulin into the cell interior that was maximal after 30 minutes of incubation. At this time point nearly 40 percent of the [125I]insulin was in the interior of the cell at a distance 1 micrometer or greater from the plasma membrane. Grain distribution and volume density analyses revealed that the intracellular insulin was concentrated in the endoplasmic reticulum and nuclear membrane.

Glucocorticoid regulation of insulin receptor gene transcription in IM-9 cultured lymphocytes.

We have reported that glucocorticoids increase steady state insulin receptor mRNA levels in target cells. In the present study using IM-9 cultured human lymphocytes, we investigated the mechanism responsible for this glucocorticoid mediated increase in insulin receptor mRNA levels. Incubation of IM-9 cells with 100 nM dexamethasone for 4 h stimulated a parallel increase in both polysomal and nuclear insulin receptor RNAs indicating that glucocorticoids did not alter the nuclear transport of insulin receptor RNA. Dexamethasone did not alter insulin receptor mRNA half life (t 1/2 = 140 +/- 20 min), indicating that glucocorticoids did not influence mRNA stability. Furthermore, the dexamethasone-induced increase in insulin receptor mRNA levels was not blocked by pretreatment of cells with cycloheximide indicating that the glucocorticoid effect was independent of new protein synthesis. When the labeled transcripts from nuclear run-off incubations were then hybridized to immobilize human insulin receptor cDNA, a three- to fourfold increase in transcriptional activity was observed. This transcriptional effect occurred before the increase in steady state insulin receptor mRNA levels and over the same range of dexamethasone concentrations. These studies indicate therefore a direct effect of glucocorticoids on insulin receptor gene transcription, and demonstrate that the insulin receptor gene is under hormonal control.

Identification of unique nuclear regulatory proteins for the insulin receptor gene, which appear during myocyte and adipocyte differentiation.

To define the molecular mechanism(s) that activate insulin receptor gene transcription during cell differentiation, we tested nuclear extracts from BC3H-1 muscle cells for their binding to the 5'-flanking region of the human insulin receptor gene. DNA binding activity of nuclear extracts was low in undifferentiated BC3H-1 cells and increased significantly during differentiation. Gel retardation assays, combined with DNase I footprinting, showed that the increased insulin receptor gene transcription occurring during differentiation was directly correlated with the appearance of DNA binding proteins that specifically interacted with two AT-rich sequences of the regulatory region of the insulin receptor gene. Fibroblast growth factor, a known inhibitor of the transcription of muscle-specific DNA binding proteins, did not inhibit the appearance of these insulin receptor DNA binding proteins. When 3T3-L1 cells differentiate from preadipocytes to adipocytes, insulin receptor gene transcription significantly increases. In differentiated adipocytes, the same two insulin receptor DNA binding proteins markedly increased. Reporter gene analysis with the two AT-rich sequences demonstrated that both of these regions of the insulin receptor gene had the characteristics of promoter rather than enhancer elements. Thus, these proteins interacting with these AT-rich sequences may have major importance in regulating the expression of the insulin receptor in target tissues.

Human diabetes associated with defects in nuclear regulatory proteins for the insulin receptor gene.

The control of gene transcription is mediated by sequence-specific DNA-binding proteins (trans-acting factors) that bind to upstream regulatory elements (cis elements). We have previously identified two DNA-binding proteins that specifically interact with two unique AT-rich sequences of the 5' regulatory region of the insulin receptor gene which have in vivo promoter activity. Herein we have investigated the expression of these DNA-binding proteins in cells from two unrelated patients with insulin resistance and non-insulin-dependent diabetes mellitus. In these patients, the insulin receptor gene was normal. In EBV-transformed lymphoblasts from both patients, insulin receptor mRNA levels and insulin receptor expression were decreased. The expression of nuclear-binding proteins for the 5' regulatory region of the insulin receptor gene was markedly reduced, and this defect paralleled the decrease in insulin receptor protein expression. These studies indicate that DNA-binding proteins to the regulatory region of the insulin receptor gene are important for expression of the insulin receptor. Further, they suggest that in affected individuals, defects in the expression of these proteins may cause decreased insulin receptor expression and insulin resistance.

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.

Dual regulation of glycogen metabolism by insulin and insulin-like growth factors in human hepatoma cells (HEP-G2). Analysis with an anti-receptor monoclonal antibody.

Insulin and the insulinlike growth factors (IGF-I and IGF-II) are members of a family of hormones that regulate the metabolism and growth of many tissues. Cultured HEP-G2 cells (a minimal deviation human hepatoma) have insulin receptors and respond to insulin by increasing their glycogen metabolism. In the present study with HEP-G2 cells, we used 125I-labeled insulin, IGF-I, and IGF-II to identify distinct receptors for each hormone by competition-inhibition studies. Unlabeled insulin was able to inhibit 125I-IGF-I binding but not 125I-IGF-II binding. A mouse monoclonal antibody to the human insulin receptor that inhibits insulin binding and blocks insulin action inhibited 75% of 125I-insulin binding, but inhibited neither 125I-IGF-I nor 125I-IGF-II binding. When glycogen metabolism was studied, insulin stimulated [3H]glucose incorporation into glycogen in a biphasic manner; one phase that was 20-30% of the maximal response occurred over 1-100 pM, and the other phase occurred over 100 pM-100 nM. The anti-receptor monoclonal antibody inhibited the first phase of insulin stimulation but not the second. Both IGF-I and IGF-II stimulated [3H]glucose incorporation over the range of 10 pM-10 nM; IGF-I was three to fivefold more potent. The monoclonal antibody, however, was without effect on IGF regulation of glycogen metabolism. Therefore, these studies indicate that insulin as well as the IGFs at physiological concentrations regulate glycogen metabolism in HEP-G2 cells. Moreover, this regulation of glycogen metabolism is mediated by both the insulin receptor and the IGF receptors.

Muscle cell differentiation is associated with increased insulin receptor biosynthesis and messenger RNA levels.

Muscle is a major tissue for insulin action. To study the effect of muscle differentiation on insulin receptors, we employed cultured mouse muscle BC3H-1 and C2 cells. In both cell lines differentiation from myoblasts to myocytes was associated with a 5-10-fold increase in specific 125I-insulin binding to intact cells. When 125I-insulin binding was carried out on solubilized myocytes and myoblasts, 125I-insulin binding to myoblasts was low. After differentiation the number of insulin receptors increased 5-10-fold. In contrast to insulin binding, insulin growth factor I receptor binding was elevated in myoblasts and was decreased by 50% in myocytes. Specific radioimmunoassay of the insulin receptor indicated that the increase in insulin binding to myocytes was due to an increase in insulin receptor content. Studies employing [35S]methionine indicated that this increase in insulin-binding sites reflected an increase in insulin receptor biosynthesis. To study insulin receptor gene expression, myoblast and myocyte mRNA was isolated and analyzed on Northern and slot blots. Differentiation from myoblasts to myocytes was accompanied by a 5-10-fold increase in insulin receptor mRNA. These studies demonstrate, therefore that differentiation in muscle cells is accompanied by increased insulin receptor biosynthesis and gene expression.

Regulation of insulin-like growth factor (IGF) I receptor expression during muscle cell differentiation. Potential autocrine role of IGF-II.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. The presence of specific, high affinity IGF receptors, as well as the expression of IGF peptides and binding proteins by muscle suggest that a significant component of IGF action in this tissue is mediated through autocrine and/or paracrine mechanisms. To explore autocrine/paracrine action of IGFs in muscle, we studied the regulation of the IGF-I receptor and the expression of IGF peptides during differentiation of the mouse BC3H-1 muscle cell line. Differentiation from myoblasts to myocytes was associated with a 60% decrease in IGF-I receptor sites determined by Scatchard analysis. Analysis of mRNA abundance and protein labeling studies indicated that the decrease in IGF-I receptor sites was associated with similar reductions in IGF-I receptor gene expression and receptor biosynthesis. IGF-II peptide gene expression was detected in myoblasts and increased 15-fold with differentiation; the increase in IGF-II gene expression preceded the decrease in IGF-I receptor gene expression. In contrast, IGF-I peptide gene expression was low in myoblasts and decreased slightly with differentiation. To explore the potential role of endogenous IGF-II in the differentiation-associated decrease in IGF-I receptor expression, we investigated the effects of IGF-II treatment in myoblasts. The addition of IGF-II to undifferentiated myoblasts resulted in downregulation of the IGF-I receptor which was associated with decreased IGF-I receptor biosynthesis and decreased IGF-I receptor mRNA abundance. These studies suggest, therefore, that IGF-I receptor expression during muscle cell differentiation may be regulated, at least in part, through autocrine production of IGF-II.

Physiological role for cholecystokinin in reducing postprandial hyperglycemia in humans.

It is known that the ingestion of glucose alone causes a greater increase in plasma glucose levels than ingestion of the same amount of glucose given with other nutrients. Since physiological plasma concentrations of cholecystokinin (CCK) prolong gastric emptying, it is proposed that after a meal, CCK may modify plasma glucose levels by delaying glucose delivery to the duodenum. To evaluate the effect of CCK on oral glucose tolerance, plasma CCK, insulin, and glucose levels and gastric emptying rates were measured in eight normal males before and after the ingestion of 60 g glucose with the simultaneous infusion of either saline or one of two doses of CCK-8 (12 or 24 pmol/kg per h). Gastric emptying rates were measured by gamma camera scintigraphy of technetium 99m sulfur colloid and plasma CCK levels were measured by a sensitive and specific bioassay. Basal CCK levels averaged 1.0 +/- 0.1 pM (mean +/- SEM, n = 8) and increased to 7.1 +/- 1.1 pM after a mixed liquid meal. After glucose ingestion, but without CCK infusion, CCK levels did not change from basal, and the gastric emptying t1/2 was 68 +/- 3 min. Plasma glucose levels increased from basal levels of 91 +/- 3.9 mg/dl to peak levels of 162 +/- 11 mg/dl and insulin levels increased from 10.7 +/- 1.8 microU/ml to peak levels of 58 +/- 11 microU/ml. After glucose ingestion, with CCK infused at 24 pmol/kg per h, plasma CCK levels increased to 8 pM and the gastric emptying t1/2 increased to 148 +/- 16 min. In concert with this delay in gastric emptying, peak glucose levels rose to only 129 +/- 17 mg% and peak insulin levels rose to only 24.2 +/- 4.2 microU/ml. With CCK at 12 pmol/kg per h, similar but less dramatic changes were seen. To demonstrate that endogenous CCK could modify the plasma glucose and insulin responses to oral glucose, oral glucose was given with 50 g of lipid containing long-chain triglycerides. This lipid increased peak CCK levels to 3.7 +/- 0.9 pM. Concomitant with this rise in CCK was a delay in gastric emptying and a lowering of plasma glucose and insulin values. To confirm that CCK reduced hyperglycemia by its effect on gastric motility, 36 g glucose was perfused directly into the duodenum through a nasal-duodenal feeding tube in four subjects. With duodenal perfusion of glucose, there was no change in plasma CCK levels, but plasma glucose levels increased from basal levels of 93+/-5 to 148+/-6 mg/dl and insulin levels rose from 10.6+/-3.5 to 29.5+/-5.2 microU/ml. When CCK was infused at 24 pmol/kg per h, neither the plasma glucose nor insulin responses to the duodenal administration of glucose were modified. Thus we conclude that CCK, in physiological concentrations, delays gastric emptying, slows the delivery of glucose to the duodenum, and reduces postprandial hyperglycemia. These data indicate, therefore, that CCK has a significant role in regulating glucose homeostasis in human.

Cholecystokinin bioactivity in human plasma. Molecular forms, responses to feeding, and relationship to gallbladder contraction.

A sensitive and specific bioassay for the measurement of cholecystokinin (CCK) in human plasma was developed to determine the molecular forms of CCK in circulation, CCK responses to feeding, and the physiologic role of CCK in gallbladder contraction. First, plasma was quantitatively extracted and concentrated with octadecylsilylsilica, and the extracts were then assayed for their ability to stimulate amylase release from isolated rat pancreatic acini. Acini were highly sensitive to CCK whereas gastrin reacted only weakly in this system. With the assay, plasma levels of cholecystokinin octapeptide (CCK-8) bioactivity as low as 0.2 pM were detectable. CCK bioactivity in plasma was inhibited by the CCK antagonist, bibutyryl cyclic guanosine monophosphate, and was eliminated by immunoadsorption with an antibody directed against the carboxyl terminus of CCK. Detection of fasting levels of CCK was possible in all individuals tested and averaged 1.0 +/- 0.2 pM (mean +/- SE, n = 22) CCK-8 equivalents. Plasma CCK biological activity was normal in patients with gastrin-secreting tumors. After being fed a mixed liquid meal, CCK levels rose within 15 min to 6.0 +/- 1.6 pM. The individual food components fat, protein, and amino acids were all potent stimulants of CCK secretion; in contrast, glucose caused a significant but smaller elevation in plasma CCK levels. Gel filtration studies identified three major forms of CCK bioactivity in human plasma: an abundant form that eluted with CCK-33, a smaller form that eluted with CCK-8, and an intermediate form that eluted between CCK-33 and CCK-8. Ultrasonic measurements of gallbladder volume indicated that this organ decreased 51% in size 30 min after feeding a mixed liquid meal. This contraction occurred coincidentally with the increase in plasma CCK levels. Next CCK-8 was infused to obtain CCK levels similar to postprandial levels. This infusion caused a decrease in gallbladder volume, similar to that seen with a meal. The present studies indicate, therefore, that CCK can be bioassayed in fasting and postprandial human plasma. These studies also suggest that CCK may be an important regulator of gallbladder contraction.

Elevated insulin receptor content in human breast cancer.

The growth of breast cancer cells is under the regulation of hormones, growth factors, and their receptors. In the present study, we have employed a new, sensitive, and specific radioimmunoassay for the direct measurement of insulin receptors in surgical specimens of breast cancers. In 159 specimens the insulin receptor content was 6.15 +/- 3.69 ng/0.1 mg protein. This value was more than sixfold higher than the mean value found in both 27 normal breast tissues obtained at total mastectomy (0.95 + 0.68, P less than 0.001) and in six normal specimens obtained from reduction mammoplasty (0.84 +/- 0.78, P less than 0.001). The insulin receptor content in breast cancer tissues was also higher than in any normal tissue investigated including liver (Pezzino, V., V. Papa, V. Trischitta, A. Brunetti, P.A. Goodman, M.K. Treutelaar, J.A. Williams, B.A. Maddux, R. Vigneri, and I.D. Goldfine, 1989. Am. J. Physiol. 257:E451-457). The insulin receptor in breast cancer retained its ability to both bind insulin and undergo insulin-induced tyrosine kinase activation. Immunostaining of the specimens revealed that the insulin receptor was present in malignant epithelial cells, but was not detected in stromal and inflammatory cells. Univariant analysis revealed that the insulin receptor content of the tumors correlated positively with tumor size (P = 0.014), histological grading (P = 0.030), and the estrogen receptor content (P = 0.035). There were no significant correlations between insulin receptor content and the age, body weight, menopausal status, and nodal involvement of the patients. These studies indicate, therefore, that the insulin receptor content is increased in breast cancers and raise the possibility that the insulin receptor may have a role in the biology of these tumors.

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.

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.

Insulin and glucagon receptors in Morris hepatomas of varying growth rates.

The binding of both insulin and glucagon to receptors in plasma membranes from five hepatomas of varying growth rates was diminished when compared to plasma membranes from normal liver. Scatchard analyses of the binding data suggested that the decrease in glucagon binding was due to a decrease in binding capacity, whereas the decrease in insulin binding was due either to a decrease in binding affinity or to site-site interactions. The decreased binding of insulin, but not of glucagon, showed a significant correlation with increasing growth rate of the tumors. These data suggest, therefore, that decreased binding of insulin to receptors could be a feature of increasing growth rate in hepatomas.