Role for membrane and secreted insulin-like growth factor-binding protein-2 in the regulation of insulin-like growth factor action in lung tumors

The insulin-like growth factors (IGFs) have been implicated in the autocrine and/or paracrine growth of a number of tumor types, including lung tumors. Importantly, insulin-like growth factor-binding proteins (IGFBPs), which both enhance and inhibit the physiological and biological actions of the IGFs, have been shown to be secreted in vitro by a wide range of tumors. In particular, IGFBP-2 is frequently produced by human tumor cells, suggesting that this protein may be an important determinant of IGF action in tumors. In the present study, we investigated IGFBP-2 effects in lung tumor cells by examining the influence of IGFBP-2 on IGF-receptor interaction and the biological actions of IGF-I and IGF-II. Affinity cross-linking studies demonstrated expression of type-I and type-II IGF receptors on small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells and the presence of abundant membrane-associated IGFBP in SCLC cells but not in NSCLC cells. An antiserum specific for IGFBP-2 was used in immunoprecipitation and immunoblotting studies which demonstrated that the membrane-associated IGFBP identified by affinity cross-linking in SCLC cells is IGFBP-2. In NSCLC cells, both IGF-I and IGF-II bound predominantly to IGF-I receptors, whereas in SCLC cells binding was principally to surface-associated IGFBP-2. SCLC cells failed to respond to IGF-I and -II stimulation in a DNA synthesis assay. For NSCLC cells, IGF-II was a more potent stimulator of DNA synthesis than IGF-I. Soluble IGFBP-2 inhibited the binding of radiolabeled IGF-I and -II to both SCLC and NSCLC cells in a concentration-dependent manner and inhibited IGF-stimulated DNA synthesis in NSCLC cells. These observations indicate that both soluble and membrane-associated IGFBP-2 compete with IGF receptors for ligand binding and, thus, are likely to be important determinants of IGF responsiveness. The findings of the present study also indicate that the type-I receptor on NSCLC cells contains a high-affinity binding site for IGF-II which presumably mediates the biological effects of IGF-II in these cells, thereby implicating IGF-II in the autocrine/paracrine growth of NSCLC.     

Modulation of the insulin-like growth factor system by chronic alcohol feeding

Insulin-like growth factor (IGF)-I is a potent anabolic agent that plays an important role in regulating muscle protein balance. Alterations in one or more of the various components of the IGF system may be in part responsible for the muscle wasting that accompanies chronic alcohol consumption. The purpose of the present study was to characterize changes in the growth hormone-IGF axis produced by chronic alcohol consumption in rats. After 8 weeks of alcohol feeding, the IGF-I concentration was decreased in plasma (31%) as well as in the liver and skeletal muscle (40-50%), compared with pair-fed control animals. In addition, alcohol consumption decreased IGF-I mRNA abundance in liver and muscle (approximately 50%). IGF-I content in duodenum and kidney, however, was not altered by alcohol feeding. Concomitantly, the relative concentration of IGF binding protein (IGFBP)-1 was increased in plasma, liver, and muscle of alcohol-fed rats, compared with control values. In contrast, no changes in the plasma concentrations of IGFBP-2, -3, or -4 were detected in alcohol-fed rats at this time point Previous studies have indicated that elevations in glucocorticoids or decreases in insulin or growth hormone might be responsible for the decrease in IGF-I and/or the increase in IGFBP-1 in other catabolic conditions. However, there was no difference in the plasma concentrations of these hormones between alcohol-fed and control animals in this study. These data indicate that chronic alcohol feeding in rats decreases IGF-I and increases IGFBP-1 in the circulation and in skeletal muscle and that these changes appear to be independent of changes in classical hormonal regulators of the IGF system. The observed alterations in the IGF system are consistent with a reduction in the anabolic actions of IGF-I induced by chronic alcohol consumption.     

Controlling insulin-like growth factor activity and the modulation of insulin-like growth factor binding protein and receptor binding

The insulin-like growth factors (IGF) and insulin perform seemingly unique roles by causing the same metabolic effect: cellular hypertrophy. Although overlapping, there are different consequences to cellular hypertrophy induced by IGF and that induced by insulin. The IGF enhance the cell hypertrophy that is requisite for cell survival, hyperplasia, and differentiation, and insulin enhances cell hypertrophy primarily as a means to increase nutrient stores. The effects of IGF and insulin are controlled by the segregation of their receptors between different cell types. A model is discussed that describes the need for three hormones (IGF-I, IGF-II, and insulin) to control nutrient partitioning. Insulin receptor localization, as well as an episodic mode of secretion, evolved to perform the short-term action of clearing excess nutrients from the circulation. In contrast, a complex and interactive set of factors ensure that maximal IGF activity occurs only when conditions are optimal for growth. A relatively invariant rate of secretion and the IGF binding proteins serve to maintain a large mutable pool of IGF. This pool exists to ensure a constant supply of IGF to maintain the basal metabolic rate and to ensure that, once a cell begins to proliferate or differentiate, adequate exposure is available to complete the process even after severe short-term physiological insults. The IGF concentrations only change in response to prolonged differences in protein and energy availabilities, environmental and body temperatures, and external stress. Also, evidence is now emerging that describes a discrete role for trace nutrients in the regulation of IGF activity. In this latter regard, zinc has the notable role of targeting IGF binding proteins to the cell surface. New data are presented showing that zinc also changes the affinity of the type 1 IGF receptor and cell-associated IGF binding proteins to optimize IGF activity.     

Peptide growth factors regulate insulin-like growth factor binding protein production by fetal rat lung fibroblasts

Insulin-like growth factor (IGF) binding proteins (IGFBPs) are expressed in fetal lung and may provide important post-translational regulation of IGF-induced mitogenesis during lung organogenesis. Because of the observation that growth factors can control cell growth through regulation of IGFBPs, we examined IGFBP production by fetal lung fibroblasts following stimulation by peptide growth factors important for fetal lung growth and development. Fetal lung fibroblasts were cultured in serum-free medium supplemented with various growth factors for up to 48 h, and IGFBPs in conditioned medium (CM) were analyzed by ligand blot and immunoblot techniques. Accumulation of CM IGFBP-3 was increased and IGFBP-2 decreased by incubation with either keratinocyte growth factor (KGF) or epidermal growth factor (EGF). The effect of these factors on IGFBP-3 accumulation increased with time but the effects of KGF on CM IGFBP-2 decreased over 48 h of incubation. CM IGFBP-4 was increased by 24 and 48 h incubation with basic fibroblast growth factor (bFGF; 2.1- and 2.7-fold increases at 24 and 48 h, respectively) and platelet-derived growth factor-BB (PDGF-BB; 4.2- and 14.9-fold increases at 24 and 48 h, respectively), and 48 h incubation with EGF (6.3-fold increase). In 48-h coincubation experiments, EGF in combination with PDGF-BB or with bFGF, and bFGF in combination with PDGF-BB, resulted in IGFBP-4 accumulations twice that expected from a summation of the effects of either growth factor alone (IGFBP-4 increased 9.8-, 4.0-, and 1.8-fold by PDGF-BB, EGF, and bFGF, respectively; and 27.1-, 37.3-, and 13.0-fold by PDGF-BB plus EGF, PDGF-BB plus bFGF, and EGF plus bFGF, respectively). These results suggest synergistic effects of these growth factors on IGFBP-4 accumulation in fetal lung fibroblast CM. Because IGFBPs are known to regulate DNA synthesis, we speculate that peptide growth factors may alter cell proliferation in fetal lung, in part through their effect on IGFBPs.     

Role of the insulin-like growth factor system in uterine function and placental development in ruminants

Maternal nutrition during pregnancy influences fetal and placental weights. The insulin-like growth factors (IGF) are also important determinants of fetal size. Furthermore, the expression of several components of the IGF system is regulated by nutrition. Effects of nutrition on fetal growth could therefore be mediated by the IGF system in the uterus and placenta. The oviductal mucosa produces IGF-I, which may influence oviductal secretions or act directly on embryonic type 1 IGF receptors. In the uterus, IGF-I mRNA is localized to the stroma surrounding the endometrial glands, which contain high concentrations of IGF type 1 receptors. Uterine IGF-I concentrations fall during pregnancy; therefore, glandular activity is more likely influenced by systemic than local IGF-I production. The IGF-II mRNA is present in both caruncles and fetal placental mesoderm, but concentrations are much higher in the latter. The actions of IGF-I and IGF-II on the endometrium and placenta are influenced by IGF-binding proteins. In the ewe, mRNAs for IGF binding protein-1 and -5 are located in the luminal and glandular epithelia, IGF binding proteins-2 and -4 are produced in the subepithelial stroma, and IGF binding protein-4 is also in the placentome capsule; IGF binding protein-3 is more widely expressed in both maternal and fetal tissues. The IGF binding proteins, therefore, form a major barrier to the passage of IGF between the fetal and maternal circulatory systems.     

Expression of vascular endothelial growth factor mRNA in non-small-cell lung carcinomas

PURPOSE: To determine the distribution of matrix metalloproteinase-1 (MMP-1) in the uveoscleral outflow pathway and other anterior segment tissues of normal human eyes. METHODS: Normal human eyes were fixed in methacarn and sectioned and immunostained using a specific polyclonal antibody to MMP-1. Immunoreactivity was visualized using diaminobenzidine. To compare the staining intensity in various tissues, the mean optical density within the ciliary body, mid-iris stroma, iris root, uveal trabecular meshwork, cornea, and sclera was determined using imaging densitometry. To determine the cellular distribution of MMP-1 in ciliary muscle, additional sections were double-immunostained using antibodies to MMP-1 and calponin. These sections were examined by confocal laser scanning microscopy. Specificity of the antibody to MMP-1 in ocular tissues was confirmed by western blot analysis with uveal tract homogenates. RESULTS: Moderate-to-strong MMP-1 immunoreactivity was observed in ciliary muscle, iris, sclera, corneal endothelium, and ciliary nonpigmented epithelium. Lighter immunoreactivity was observed in corneal epithelium, blood vessels, trabecular meshwork, Schlemm's canal, and associated collector channels. Confocal microscopy showed that ciliary muscle MMP-1 was primarily inside ciliary muscle cells. Densitometry showed that net optical density was approximately fivefold greater in ciliary muscle, iris root, and sclera than in trabecular meshwork. CONCLUSIONS: MMP-1 was prominently identified in regions of the anterior segment of normal human eyes associated with the uveoscleral outflow pathway and in the iris, corneal endothelium, and ciliary nonpigmented epithelium. These data support the hypothesis that MMP-1 activity is involved in regulating uveoscleral outflow facility.     

The mouse intraovarian insulin-like growth factor I system: departures from the rat paradigm

Although the rat intraovarian insulin-like growth factor I (IGF-I) system is well documented, the increasing availability of null mouse mutants for components of the IGF system necessitates characterization of the mouse model as well. Therefore, we undertook to define the components of the mouse intraovarian IGF-I system and to examine its operational characteristics. The cellular pattern of ovarian gene expression was comparable in the immature rat and mouse for IGF-I and the type I IGF receptor. In both species, IGF-I messenger RNA (mRNA) is selectively expressed by granulosa cells in growing, healthy appearing follicles. Type I IGF receptor mRNA was also concentrated in granulosa cells, but was uniformly expressed in all follicles large and small, healthy and atretic appearing alike. Cellular patterns of IGF-binding protein (IGFBP) gene expression were similar in mouse and rat, except in the case of IGFBP-2. IGFBP-2 mRNA was localized to the mouse granulosa cell, in contrast to its concentration in the rat thecal-interstitial compartment. This difference in IGFBP expression pattern was also noted in cultured mouse and rat granulosa cells. Although immunoreactive IGFBP-4 (24 and 28 kDa) and IGFBP-5 (29 kDa) were shared by both species, the cultured mouse granulosa cell also featured immunoreactive IGFBP-2 (30 kDa). The mouse paradigm further differed from its rat counterpart in that a maximal dose of FSH, previously shown to suppress the elaboration of rat granulosa cell-derived IGFBPs, was without effect. The addition of IGF-I proved stimulatory to the accumulation of the 28- to 29-kDa IGFBPs, as previously reported for the rat. However, IGF-I proved inhibitory to the accumulation of the 24-kDa IGFBP (presumptive nonglycosylated IGFBP-4); no consistent effect was reported for the rat model. Functional comparisons of mouse and rat ovarian cell cultures revealed qualitatively comparable FSH-stimulated steroidogenesis, disposition of radiolabeled pregnenolone, IGF-I-amplified FSH action, and IGFBP-mediated antigonadotropic activity. These findings indicate that the mouse intrafollicular IGF-I system differs from the rat paradigm in both the makeup and regulation of granulosa cell-derived IGFBPs as well as in the intensity and character of the steroidogenic process. Studies employing the mouse model must take into account these important distinctions relative to the more established rat paradigm.     

Pregnancy-specific alterations in the expression of the insulin-like growth factor system during early placental development in the ewe

The placenta is recognized as an important determinant of fetal growth rate, yet the factors regulating its proliferation remain poorly understood. Components of the insulin-like growth factor (IGF) system were localized in the ovine uterus using in situ hybridization between days 13-55 of gestation, the period of implantation and placentome formation. IGF-II messenger RNA (mRNA) expression was intense in the fetal mesoderm, particularly at the tips of the invading placentome villi. Moderate levels of IGF-II mRNA were also observed in the maternal caruncular stroma. In contrast, expression of IGF-1 mRNA was low (compared to estrous levels) and ubiquitous decreasing as gestation advanced. IGF-binding protein-2 (IGFBP-2 mRNA was not detected until day 29 of gestation, when it appeared restricted to the dense caruncular-like stroma lining the luminal epithelium, colocalized with IGFBP-4. High concentrations of IGFBP-4 mRNA expression were also found in the placentome capsule. IGFBP-3 mRNA expression was intense in the luminal epithelium between days 13-15 of gestation. Subsequently, levels in this region dropped significantly (P < 0.001). IGFBP-3 mRNA expression was also high in the maternal placentome villi, where photographic emulsions localized expression to blood vessel walls. Peak expression of IGF type 1 receptor (IGF-1R) mRNA was found in the deep uterine glands, with intermediate expression in the superficial uterine glands. Moderate expression of IGF-1R mRNA was initially recorded in caruncular stroma, but levels in this region decreased significantly (P < 0.001) to below the detection limit of the technique after interdigitation by the fetal allantochorion. Furthermore, IGF-1R mRNA could not be detected in any fetal placentome tissue. This study, therefore, has established the pattern of expression of the IGFs, IGF-1R, and three of the IGFBPs during establishment of the ovine placenta. It will form the basis for future work to investigate how this system is regulated and to determine the role of the IGFs in placental development.     

Expression of insulin-like growth factor binding protein-4 and -5 mRNAs in adult rat forebrain

Accumulating evidence indicates that the insulin-like growth factors (IGFs) can act as neurotrophic factors. A family of at least six IGF binding proteins (IGFBPs) has been characterized. The IGFBPs prolong the half-life of IGFs in plasma and may modulate IGF action in a cell- or tissue-specific fashion. Two recently characterized IGFBPs, IGFBP-4 and -5, have been shown by northern blot hybridization to be expressed in rat brain, but their cellular sites of synthesis are poorly characterized. Because IGFBP-4 and IGFBP-5 could potentially modulate IGF actions in the brain, we used in situ hybridization histochemistry and 35S-labeled IGFBP-4 and IGFBP-5 riboprobes to localize sites of IGFBP-4 and -5 mRNA expression in adult rat brain. The two IGFBP mRNAs are abundantly expressed within discrete regions of brain. The expression patterns of the two genes are largely nonoverlapping. Notably, IGFBP-4 mRNA is highly expressed within hippocampal and cortical areas, whereas IGFBP-5 mRNA is not detected above background in these areas. Within the hippocampus, abundant IGFBP-4 mRNA expression is detected in pyramidal neurons of the subfields of Ammon's horn and the subiculum and in the granule cell layer of the anterior hippocampal continuation. In the cortex, IGFBP-4 mRNA is widely expressed in most areas and layers. In contrast, IGFBP-5, but not IGFBP-4, mRNA is detected within thalamic nuclei, leptomeninges, and perivascular sheaths. The distinct expression patterns of IGFBP-4 and -5 mRNAs within the brain suggest that these IGFBPs may modulate paracrine/autocrine actions of the IGFs in discrete brain regions or compartmentalization of the IGFs within the brain.     

Developmental pattern of fetal growth hormone, insulin-like growth factor I, growth hormone binding protein and insulin-like growth factor binding protein-3

AIMS: To evaluate the developmental pattern of fetal growth hormone (GH), insulin-like growth factor I (IGF-I), GH binding protein (GHBP) and IGF binding protein-3 (IGF-3); to determine the implications for fetal growth. METHODS: Serum GH, IGF-I, GHBP and IGFBP-3 were measured in 53 fetuses, 41 aged 20-26 weeks (group A) and 12 aged 31-38 weeks (group B). Fetal blood samples were obtained by direct puncture of the umbilical vein in utero. Fetal blood samples were taken to rule out beta thalassaemia, chromosome alterations, mother to fetus transmissible infections, and for maternal rhesus factor. GHBP was determined by gel filtration chromatography of serum incubated overnight with 125I-GH. GH, IGF-I and IGFBP-3 were determined by radioimmunoassay. RESULTS: Fetal serum GH concentrations in group A (median 29 micrograms/l, range 11-92) were significantly higher (P < 0.01) than those of group B (median 16.7 micrograms/l, range 4.5-29). IGF-I in group A (median 20 micrograms/l, range 4.1-53.3) was significantly lower (P < 0.01) than in group B (median 75.2 micrograms/l, range 27.8-122.3). Similarly, IGFBP-3 concentrations in group A (median 950 micrograms/l, range 580-1260) were significantly lower than those of group B (median 1920 micrograms/l, range 1070-1770). There was no significant difference between GHBP values in group A (median 8.6%, range 6.6-12.6) and group B (median 8.3%, range 6-14.3). Gestational age correlated positively with IGF-I concentrations (P < 0.0001) and IGFBP-3 (P < 0.0001) and negatively with GH (P < 0.0001). GHBP values did not correlate with gestational age. Multiple regression analysis showed a negative correlation between GH:IGF-I ratio and fetal growth indices CONCLUSIONS: The simultaneous evaluation of fetal GH, IGF-I, IGFBP-3 and GHBP suggests that the GH-IGF-I axis might already be functional in utero. The progressive improvement in the efficiency of this axis in the last part of gestation does not seem to be due to an increase in GH receptors.     

Preparation of N epsilon B28-monoazidobenzoyl insulin-like growth factor I and photoaffinity labeling of insulin-like growth factor I receptor

Recombinant human insulin-like growth factor I (hIGF-I) was reacted with azidobenzoyl hydroxysuccinimide to produce a mixture of photoactive hIGF-I derivatives. The mixture was purified by reversed-phase HPLC to yield three mono-substituted azidobenzoyl hIGF-Is. One of the derivatives was identified by amino acid sequencing as N epsilon B28-monoazidobenzoyl hIGF-I. This derivative was indistinguishable from native hIGF-I when bioassayed in Rat-1 fibroblasts. A 120-kDa band, the alpha subunit of the IGF-I receptor, was specifically labeled in Rat-1 plasma membranes by this photoprobe. The labeling of this band was reduced by hIGF-I at 1 nM and completely abolished by hIGF-I, but not insulin, at 100 nM, indicating the specificity of the photolabeling of the IGF-I receptor by this fully active IGF-I photoprobe.     

Selective expression of insulin-like growth factor II in the songbird brain

Neuronal replacement occurs in the forebrain of juvenile and adult songbirds. To address the molecular processes that govern this replacement, we cloned the zebra finch insulin-like growth factor II (IGF-II) cDNA, a factor known to regulate neuronal development and survival in other systems, and examined its expression pattern by in situ hybridization and immunocytochemistry in juvenile and adult songbird brains. The highest levels of IGF-II mRNA expression occurred in three nuclei of the song system: in the high vocal center (HVC), in the medial magnocellular nucleus of the neostriatum (mMAN), which projects to HVC, and to a lesser extent in the robust nucleus of the archistriatum (RA), which receives projections from HVC. IGF-II mRNA expression was developmentally regulated in zebra finches. In canary HVC, monthly changes in IGF-II mRNA expression covaried with previously reported monthly differences in neuron incorporation. Combining retrograde tracers with in situ hybridization and immunocytochemistry, we determined that the HVC neurons that project to area X synthesize the IGF-II mRNA, whereas the adjacent RA-projecting neurons accumulate the IGF-II peptide. Our findings raise the possibility that within HVC IGF-II acts as a paracrine signal between nonreplaceable area X-projecting neurons and replaceable RA-projecting neurons, a mode of action that is compatible with the involvement of IGF-II with the replacement of neurons. Additional roles for IGF-II expression in songbird brain are likely, because expression also occurs in some brain areas outside the song system, among them the cerebellar Purkinje cells in which neurogenesis is not known to occur.     

Biological characterization of human epithelial ovarian carcinoma cells in primary culture: the insulin-like growth factor system

Current hypotheses for explaining which sex cares for offspring assume a relationship between the mode of fertilization and the sex showing parental care. In general, it is hypothesized that maternal care should be concentrated in taxa with internal fertilization, and paternal care should be concentrated in taxa with external fertilization. Studies have supported this relationship; however, new comparative techniques and new data on parental care and the frequency of internal fertilization in anurans suggest that the relationship should be re-evaluated. I examined the relationship between mode of fertilization and sex providing parental care in 334 taxa of anurans using concentrated changes tests and in 396 taxa of anurans using a method developed by Ridley (1983. The Explanation of Organic Diversity. The Comparative Method and Adaptations of Mating. Oxford: Clarendon). The results of the concentrated changes tests showed that both female and male parental care are randomly distributed among taxa with respect to mode of fertilization. However, using Ridley's method, I found significant relationships between mode of fertilization and sex providing parental care. The observed and expected numbers of transitions from external fertilization and no parental care to external fertilization and male parental care or to internal fertilization and female parental care are not qualitatively different. Therefore, the results of my analyses suggest that current hypotheses for explaining the occurrence of maternal versus paternal care in anurans should be reconsidered. Copyright 1998 The Association for the Study of Animal Behaviour.     

Role of milk-borne vs endogenous insulin-like growth factor I in neonatal growth

Neonatal pigs are characterized by a high efficiency of nutrient utilization and rapid growth rate. The utilization of dietary protein for lean tissue growth is particularly efficient in neonatal pigs and is associated with a high rate of skeletal muscle protein synthesis and deposition. In support of these high growth rates, neonatal pigs consume a milk diet that has a high biological value and is abundant in growth factors, including insulin and IGF-I. During the neonatal period, there are developmental changes in the circulating concentrations of, and tissue responsiveness to, hormones, particularly insulin, IGF-I, and growth hormone that play a central role in growth regulation. Our goal has been to characterize the dietary factors and specific aspects of endocrine function that are responsible for the anabolic stimulus that helps to sustain the high rates of protein deposition in neonatal pigs. Our results suggest that, despite the abundance of growth factors in milk and colostrum, the intake of nutrients is the primary anabolic stimulus for protein synthesis and this response declines with age. There is, however, a nonnutritive and as-yet-unidentified component in colostrum that provides a specific anabolic stimulus for skeletal muscle in newborns, but this is probably neither insulin nor IGF-I. Our studies also indicate that circulating concentration of IGF-I are not a primary stimulus of skeletal muscle protein synthesis and that the primary endocrine signal that mediates the response to nutrient intake may be insulin. Future research should address how the local expression of IGF and the function of insulin and IGF receptors affect the responsiveness of anabolic processes to nutrient intake and hence the efficiency of neonatal growth.     

Structure and function of the insulin-like growth factor I receptor

Two manipulations are argued to distinguish between instance-based and abstract rule-based accounts of invariant learning. Three experiments examined the effects of manipulating the type of invariant feature in the learning set, and the type of training schedules prior to test. In line with traditional research, selection bias at test was present when the invariant was the consistent inclusion of a stimulus item in the learning set. However, the degree of bias was identical when the invariant was the consistent exclusion of the stimulus item. In addition, negative transfer of training was observed when subjects were trained on one learning set and then shifted training to the opposite learning set, but no positive transfer of training was observed when subjects were trained on one learning set and then continued training using the same learning set. These results are argued to be evidence for instance-based accounts of invariant learning.     

Improvement of erectile function in diabetic rats by insulin: possible role of the insulin-like growth factor system

Erectile dysfunction is commonly experienced in men with diabetes mellitus. We report that the intracavernous pressure (ICP) rise in diabetic rats was 55% of the control and returned to normal following insulin (I) or insulin plus free oxygen scavenger (I + S) treatment. Insulin-like growth factor (IGF) binding protein (IGFBP) -3, -4, and -5 messenger RNA (mRNA) levels in the major pelvic ganglia (MPG) of diabetic rats were elevated by 2-fold, 2.6-fold, and 2.5-fold, respectively. Both I and I + S returned IGFBP-4 and 5 mRNA levels to normal, whereas IGFBP-3 gene expression was severely inhibited. IGFBP-2 gene expression was greatly inhibited by diabetes and was unresponsive to treatment. In the penis of diabetic rats, IGFBP-2 and -4 mRNA levels were low, whereas IGFBP-3 mRNA levels were elevated 10-fold. These effects were reversed by I and I + S. I and I + S also corrected the IGFBP-3 expression pattern. IGF-I gene expression in the penis and MPG was not significantly increased (P < 0.05) by diabetes and returned to normal levels following I or I + S treatment. Because IGFs are potent regulatory factors in vascular tone, this newly described activity of insulin may play an important role in the improvement of erectile function seen clinically and in animal models.