Insulin Binding Protein Research Articles

Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP‐1c

Steatosis is an accumulation of triglycerides in the liver. Although an excessive availability of plasma fatty acids is an important determinant of steatosis, lipid synthesis from glucose (lipogenesis) is now also considered as an important contributing factor. Lipogenesis is an insulin- and glucose-dependent process that is under the control of specific transcription factors, sterol regulatory element binding protein 1c (SREBP-1c), activated by insulin and carbohydrate response element binding protein (ChREBP) activated by glucose. Insulin induces the maturation of SREBP-1c by a proteolytic mechanism initiated in the endoplasmic reticulum (ER). SREBP-1c in turn activates glycolytic gene expression, allowing glucose metabolism, and lipogenic genes in conjunction with ChREBP. Lipogenesis activation in the liver of obese markedly insulin-resistant steatotic rodents is then paradoxical. Recent data suggest that the activation of SREBP-1c and thus of lipogenesis is secondary in the steatotic liver to an ER stress. The ER stress activates the cleavage of SREBP-1c independent of insulin, thus explaining the paradoxical stimulation of lipogenesis in an insulin-resistant liver. Inhibition of the ER stress in obese rodents decreases SREBP-1c activation and lipogenesis and improves markedly hepatic steatosis and insulin sensitivity. ER is thus a new partner in steatosis and metabolic syndrome which is worth considering as a potential therapeutic target.

Insulin-Binding Canavalin Is Present in Canavalia ensiformis Seed Coat

An insulin-binding protein was isolated from Canavalia ensiformis seed coat, by using an insulin-Sepharose 4B affinity chromatography, and the protein was identified as canavalin (Canavalia 7S globulin) by mass spectrometry analysis. The major novelty of these data is the acidic nature of this globulin insulin-binding, in contrast to the basic Bg-like insulin-binding proteins so far reported in plants.

Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance

BackgroundInsulin and insulin-like growth factors (IGFs) signal through a highly conserved pathway and control growth and metabolism in both vertebrates and invertebrates. In mammals, insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and modulate their mitogenic, anti-apoptotic and metabolic actions, but no functional homologs have been identified in invertebrates so far.ResultsHere, we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2) binds Drosophila insulin-like peptide 2 (Dilp2) and inhibits growth non-autonomously. Whereas over-expressing Imp-L2 strongly reduces size, loss of Imp-L2 function results in an increased body size. Imp-L2 is both necessary and sufficient to compensate Dilp2-induced hyperinsulinemia in vivo. Under starvation conditions, Imp-L2 is essential for proper dampening of insulin signaling and larval survival.ConclusionImp-L2, the first functionally characterized insulin-binding protein in invertebrates, serves as a nutritionally controlled suppressor of insulin-mediated growth in Drosophila. Given that Imp-L2 and the human tumor suppressor IGFBP-7 show sequence homology in their carboxy-terminal immunoglobulin-like domains, we suggest that their common precursor was an ancestral insulin-binding protein.

Characterization of differential gene expression profiles in diabetic embryopathy using DNA microarray analysis

The molecular mechanisms by which maternal diabetes impairs embryogenesis are not established. This study aimed to determine the developmental genes and molecular pathways that are involved in diabetic embryopathy, by comparing gene expression profiles in the yolk sacs between the embryos of diabetic and control rats by using DNA microarray analysis. Diabetes was induced in female rats by injecting streptozotocin (65 mg/kg) intravenously. Glucose levels were controlled by subcutaneously implanting insulin pellets. The female rats were mated with normal male rats. At gestation day 4, the insulin pellets were removed from a group of animals, making them hyperglycemic. The animals with insulin pellets served as controls. At gestational day 12, embryos were explanted, and yolk sacs were collected. Total RNA, free of DNA contamination, was extracted from the yolk sacs. Complementary DNA probes were synthesized, labeled with Cy3 and Cy5 fluorescent dyes, and used to hybridize rat oligo-array containing 10,000 genes. Data were analyzed by using 1-sample t test on log2 ratios, with P < .05 representing a significant difference. The changes in expression levels of important genes were verified with the use of a real-time polymerase chain reaction (PCR). Five microarray experiments produced consistent results. A total of 101 genes were found to be differentially expressed between the embryos of diabetic and control rats. Analyses that used PathwayAssist (Ariadne Genomics, Rockville, MD) revealed a number of potential signaling pathways and genes involved in insulin signaling and stress response (insulin 2, insulin-binding protein 1, GST pi1), cell growth (GAP43, CSF1R, HGF), calcium signaling (calbindin 3, CBP A6), and PKC signaling (PKCBP beta15, FABP5), in concert with prior biochemical and molecular findings. These observations show significant alterations in expression of developmental and stress response genes in diabetic embryopathy, and demonstrate, for the first time, that the yolk sac cells express insulin during early development. In addition, these data also demonstrate that hyperglycemia induces altered gene expression, resulting in aberrant cell signaling, morphogenesis, and embryopathy.

A piezoelectric micro-cantilever bio-sensor using the mass-micro-balancing technique with self-excitation

A biosensor was developed for using in a Lab-On-a-Chip (LOC). The sensor detects the change in the resonance frequency of a micro-cantilever with a piezoelectric film. This is the mass micro-balancing technique, which has been successfully used for detecting bio-materials in the quartz crystal microbalance (QCM). The PZT film, a piezoelectric film, is designed to act as both sensor and actuator. The geometry of the micro cantilever is optimized to maximize the sensitivity and minimize the environmental effects such as viscous damping and added mass effect in liquid. The fabricated sensor is composed of a 100 μm long, 30 μm wide, and 5 μm thick cantilever with a 2.5 μm thick piezoelectric (PZT) layer on it. The ratio of thickness to length of the micro cantilever is very high compared to others in micro cantilever-based studies. This high aspect ratio is the key to maximize the sensitivity and minimize the environmental effects. The fabricated micro sensor was tested by detecting the mussel gluing protein, the insulin-anti insulin binding protein and the poly T-sequence DNA.

A self-excited micro cantilever biosensor actuated by PZT using the mass micro balancing technique

A micro biosensor, which can be applied to a Lab-On-a-Chip (LOC), is developed in order to detect biomaterials such as protein or DNA. The biomaterials are detected by mass micro-balancing technique, which measures the change of the resonant frequency of the sensor structure. The sensor structure consists of a micro cantilever actuated by piezoelectric PZT film. The PZT film is designed to act as both a sensor and an actuator. The geometry of the micro cantilever is determined so as to maximize the sensitivity of the sensor, and environmental effects such as added mass effect in liquid are also considered in the structural analysis. The micro cantilever is 100 μm in length, 30 μm in width and 5 μm in thickness, and the PZT film thickness and length are 2.5 and 50 μm, respectively. The first resonant frequency of the PZT micro cantilever is 1.2 ∼ 1.3 MHz. Lastly the fabricated micro-biosensor using the self-excited PZT-micro cantilever is tested by detecting the human insulin-anti human insulin binding protein, the poly T-sequence DNA, the K20-Thiol DNA and the K40-Thiol DNA in air.

Bone mass and turnover in women with epilepsy on antiepileptic drug monotherapy

Antiepileptic drugs, particularly cytochrome P450 enzyme inducers, are associated with disorders of bone metabolism. We studied premenopausal women with epilepsy receiving antiepileptic drug monotherapy (phenytoin, carbamazepine, valproate, and lamotrigine). Subjects completed exercise and nutrition questionnaires and bone mineral density studies. Serum was analyzed for indices of bone metabolism including calcium, 25-hydroxyvitamin D, parathyroid hormone, insulin growth factor I, insulin binding protein III, and bone formation markers, bone-specific alkaline phosphatase, and osteocalcin. Urine was analyzed for cross-linked N-telopeptide of type I collagen, a bone resorption marker. Calcium concentrations were significantly less in subjects receiving carbamazepine, phenytoin, and valproate than in those receiving lamotrigine (p = 0.008). Insulin growth factor-I was significantly reduced in subjects receiving phenytoin compared with those receiving lamotrigine (p = 0.017). Subjects receiving phenytoin had significantly greater levels of bone-specific alkaline phosphatase (p = 0.007). Our results demonstrate that phenytoin is associated with changes in bone metabolism and increased bone turnover. The lower calcium concentrations in subjects taking carbamazepine or valproate compared with those taking other antiepileptic drugs suggest that these antiepileptic drugs may have long-term effects. Subjects receiving lamotrigine had no significant reductions in calcium or increases in markers of bone turnover, suggesting this agent is less likely to have long-term adverse effects on bone.

Adrenalectomy Does Not Block the Inhibition of Mammary Carcinogenesis by Dietary Energy Restriction in Rats

Dietary energy restriction (DER) has been shown to reproducibly inhibit chemically induced mammary carcinogenesis. The inhibitory activity of DER has been reported to be associated with an increase in circulating corticosterone as well as a decrease in insulin-like growth factor 1 (IGF-1). To determine whether the adrenal glands are required for cancer inhibitory activity, the effects of DER were investigated in adrenalectomized (ADX) rats. Female Sprague-Dawley rats, 29-31 per group, were injected with 0.05 g 1-methyl-1-nitrosourea/kg body wt at 21 d of age, sham operated (SHAM) or bilaterally ADX at 24 d of age, and after 3 d adapted to meal feeding during which rats ate ad libitum (AL) or were restricted to 60% of AL energy intake. ADX resulted in a marked reduction in serum corticosterone in both AL and DER rats. Whereas the carcinogenic response in the mammary gland was not statistically different in SHAM-AL and ADX-AL rats, ADX did not block the cancer inhibitory activity of DER. In fact, cancer inhibitory activity was greatest in ADX-DER rats. Circulating levels of glucose, insulin, IGF-1, and IGF binding protein 3 also were reduced in DER rats. Collectively, these findings indicate that adrenal glands are not required for manifestation of the cancer inhibitory activity of DER. If circulation-borne factors such as corticosterone or IGF-1 are involved in the inhibition of mammary carcinogenesis by DER, IGF-1 is likely to play a greater role than corticosterone.

Insulin inhibition of protein degradation in cells expressing wild-type and mutant insulin receptors.

The mechanism by which insulin decreases protein degradation is unknown. We examined insulin binding and degradation (125I[A14]insulin) and protein degradation (3H-leucine labeling) in Chinese hamster ovary (CHO) cells transfected with wild-type (WI) and mutant human insulin receptors. The deltaExon-16 mutant is missing the juxtamembrane domain that mediates endocytosis. The delta343 mutant receptor lacks the tyrosine kinase structural domain but retains the juxtamembrane internalization domain. The mutant deltaNPEY lacks the single NPEY sequence located 16 residues after the end of the transmembrane domain. Null transfected cells (NEO) not expressing human receptors were studied as controls. The WT and deltaNPEY cells equivalently internalized and degraded insulin; delta343 cells internalized and degraded insulin, but at a reduced rate; deltaExon-16 cells internalized and degraded significantly less insulin than the other mutants; NEO cells showed essentially no internalization and degradation. In contrast, all cell types showed the same efficacy at inhibition of protein degradation, albeit at different potencies. These results suggest insulin actions are mediated by multiple and redundant effector systems, but that receptor tyrosine kinase activity is not required for inhibition of protein degradation.

Luteal phase deficiency in recreational runners: evidence for a hypometabolic state.

Exercising women with amenorrhea exhibit a hypometabolic state. The purpose of this study was to evaluate the relationship of luteal phase deficient (LPD) menstrual cycles to metabolic hormones, including thyroid, insulin, human GH (hGH), leptin, and IGF-I and its binding protein levels in recreational runners. Menstrual cycle status was determined for three consecutive cycles in sedentary and moderately active women. Menstrual status was defined as ovulatory or LPD. Subjects were either sedentary (n = 10) or moderately active (n = 20) and were matched for age (27.7 +/- 1.2 yr), body mass (60.2 +/- 3.3 kg), menstrual cycle length (28.4 +/- 0.9 d), and reproductive age (14.4 +/- 1.2 yr). Daily urine samples for the determination of estrone conjugates, pregnanediol 3-glucuronide, and urinary levels of LH were collected. Blood was collected on a single day during the follicular phase (d 2-6) of each menstrual cycle for analysis of TSH, insulin, total T3, total T4, free T4, leptin, hGH, IGF-I, and IGF binding protein (IGFBP)-1 and IGFBP-3. Among the 10 sedentary subjects, 28 of 31 menstrual cycles were categorized as ovulatory (SedOvul). Among the 20 exercising subjects, 24 menstrual cycles were included in the ovulatory category (ExOvul), and 21 menstrual cycles were included in the LPD category (ExLPD). TSH, total T4, and free T4 levels were not significantly different among the three categories of cycles. Total T3 was suppressed (P = 0.035) in the ExLPD (1.63 +/- 0.07 nmol/liter) and the ExOvul categories of cycles (1.75 +/- 0.8 nmol/liter) compared with the SedOvul category of cycles (2.15 +/- 0.1 nmol/liter). Leptin levels were lower (P < 0.001) in both the ExOvul (5.2 +/- 0.4 microg/liter) and the ExLPD categories of cycles (5.1 +/- 0.4 microg/liter) when compared with the SedOvul category of cycles (13.7 +/- 1.7 microg/liter). Insulin was lower (P = 0.009) only in the ExLPD category of cycles (31.9 +/- 2.8 pmol/liter) compared with the SedOvul (60.4 +/- 8.3 pmol/liter) and ExOvul (61.8 +/- 10.4 pmol/liter) categories of cycles. IGF-I, IGFBP-1, IGFBP-3, IGF-I/IGFBP-1, IGF-I/IGFBP-3, and hGH were comparable among the different categories of cycles. These data suggest that exercising women with LPD menstrual cycles exhibit hormonal alterations consistent with a hypometabolic state that is similar to that observed in amenorrheic athletes and other energy-deprived states, although not as comprehensive. These alterations may represent a metabolic adaptation to an intermittent short-term negative energy balance.

Insulin inhibits peroxisomal fatty acid oxidation in isolated rat hepatocytes.

Inhibition by insulin of long chain fatty acid oxidation in mitochondria is mediated in part by elevating malonyl-CoA levels, which inhibit carnitine palmitoyl-transferase I. Whether insulin alters peroxisomal oxidation has not been studied. We present data which show that insulin inhibits the oxidation of palmitic acid by peroxisomes (IC(50) = 8.5 x 10(-11) M) at hormone concentrations 100-fold less than those needed for mitochondrial inhibition (IC(50) = 1.3 x 10(-8) M). We used a purified peroxisome preparation to study the mechanism of insulin action. Insulin had a direct effect in the peroxisome preparations to decrease oxygen consumption, fatty acyl-CoA oxidizing system activity and acyl-CoA oxidase by approximately 40%, 30% and 15%, respectively. Since insulin degrading enzyme (IDE) is an insulin-binding protein known to be in peroxisomes, we studied the effect of an inhibitory anti-IDE antibody on the ability of insulin to inhibit the fatty acyl-CoA oxidizing system. The antibody eliminated the inhibitory effect of insulin. We conclude that insulin inhibits peroxisomal fatty acid oxidation by a mechanism requiring IDE.

Cord Blood Leptin and Insulin-Like Growth Factor Levels are Independent Predictors of Fetal Growth

The insulin-like growth factor (IGF) system is the dominant endocrine regulator of fetal growth, whereas insulin has a permissive role. Although a role for leptin in fetal growth has been suggested recently, the mechanism by which leptin may be related to fetal growth is not known; but leptin may interact with the IGF system in utero as it does in the extrauterine life. In the context of a hospital-based case control study, we collected anthropometric and demographic data and measured serum leptin, IGF-I, IGF-II, insulin, cortisol, and IGF binding protein 3 concentrations in 142 cord blood samples from full-term deliveries. Cord leptin, IGF-I, and insulin levels correlated positively with birth weight (r = 0.46, r = 0.41, and r = 0.21, respectively, P < 0.01) by univariate analysis and were significantly higher in large-for-gestational-age (LGA) infants, compared with appropriate-for-gestational-age (AGA) infants. Cord leptin concentrations correlated with insulin levels (r = 0.36, P<0.01) but not with IGF-I levels (r = 0.20). Multiple linear and logistic regression analysis demonstrated an independent positive relationship of both leptin and IGF-I with birth weight and AGA/LGA status. The positive association of leptin levels with birth weight and AGA/LGA status cannot be attributed to IGF-I. This suggests the existence of alternative mechanisms underlying leptin’s associations with fetal growth that should be further explored.

Constitutive Secretion of an Endogenous Insulin-like Peptide Binding Protein with High Affinity for Insulin in Spodoptera frugiperda (Sf9) Cell Cultures

Serum-free medium from batch cultures of Sf9 insect cells was examined for the occurrence of proteins related to the insulin-like growth factor family. We found that the Sf9 cell line constitutively produced and secreted a soluble protein with a MW of 27 kDa that exerted specific binding to human insulin-like growth factor-I (IGF-I) and -II. Moreover, the secreted protein bound human insulin and human proinsulin with higher affinity than IGF-I and -II. The order of affinity to the insulin peptides, determined by competitive inhibition of ligand binding, was: insulin > proinsulin > IGF-I ⪢ IGF-II. The dissociation constant (kd) for IGF-II was 28.5 ± 1.7 nM and for insulin 7.2 ± 1.3 nM, as determined by Scatchard plot analysis. The results suggest that the Sf9 cells produce an insulin binding protein similar to the human insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1).

Is growth hormone hypersecretion in diabetic adolescen_t girls also a daytime problem?

Glycaemic control often deteriorates during puberty in girls with insulin dependent diabetes mellitus (IDDM). This may be due in part to the normal psychosocial changes associated with adolescence. Puberty is, however, also characterized by rapid somatic development, orchestrated by hormonal changes. Some of these hormones play a major role in glucose homeostasis. We have examined the insulin-GH-IGF-I axis in 11 adolescent girls with poorly controlled insulin dependent diabetes and compared the data with those of 10 non-diabetic girls matched for age, pubertal stage and body mass index (BMI). Serum profiles of glucose, insulin, GH and IGF binding protein 1 (IGFBP1) were analysed in addition to IGF-I in serum and nocturnal urinary excretion of GH. Serum glucose, insulin and IGFBP1 were measured every hour for 24 h, whereas GH in serum was measured every 30 minutes during the same period. Nocturnal urinary GH was analysed as a mean of three consecutive nights. The insulin profiles of the IDDM patients were flat with low post-prandial peaks, corresponding to only one-third of the peaks of the non-diabetic girls. The integrated insulin levels, both during 24-h sampling and during daytime, were significantly lower in the diabetic group. There were no differences during night-time. The diabetic patients had elevated mean baseline levels of serum GH (IDDM 2.8 +/- 0.5 mU/l, controls 0.7 +/- 0.2; P < 0.001), a higher 24-h mean serum GH level (9.8 +/- 1.7 mU/l vs. 4.4 +/- 0.7; P < 0.001), significantly more peaks and a urinary GH excretion twice as high as in the non-diabetic group. An interesting observation was the finding of marked differences in daytime GH concentrations between the groups, both regarding overall integrated levels (GH AUC 103 +/- 15.8 and 35.9 +/- 7.1 mU/l x 12 h, respectively; P < 0.005) as well as baseline levels (3.8 +/- 0.6 mU/l vs. 0.7 +/- 0.2; P < 0.001). In contrast, during night-time only the mean basal levels of GH differed. The level of IGF-I was reduced in the diabetic group compared with the healthy controls (IDDM 233 +/- 19 micrograms/l vs. controls 327 +/- 21; P < 0.005). In addition, the IDDM patients had significantly increased concentrations of IGFBP 1, but kept a normal diurnal rhythm with a pronounced night peak. Hypoinsulinaemia in adolescent IDDM patients, particularly in the portal hepatic circulation, results in decreased IGF-I and increased IGFBP 1 production in the liver. High levels of IGFBP 1 may, in turn, reduce the bioactivity of IGF-I even further. Low levels of IGF-I will lead to increased GH secretion. Earlier studies on the relationship between GH and diabetic control have focused on elevated GH levels during the night. In this study we have observed markedly elevated levels of GH also during daytime in adolescent IDDM patients. This indicates increased insulin resistance and insulin demand also during the day in diabetic subjects. The increased insulin resistance may result in hyperglycaemia leading to additional insulin resistance. A vicious circle may thus be induced, accelerating metabolic impairment in poorly controlled adolescent IDDM girls.

Insulin signaling in the yeast Saccharomyces cerevisiae. 3. Induction of protein phosphorylation by human insulin.

A low affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae has been identified recently (Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W., preceding paper in this issue). Since the mammalian insulin receptor functions as a tyrosine kinase with autophosphorylation capacity, kinase studies were performed with the partially purified insulin-binding protein preparation. Incubation with [gamma-32P]ATP in vitro led to phosphorylation of the 53-kDa insulin-binding protein on serine but not on tyrosine residues. In addition, a 70-kDa polypeptide, copurified with the insulin-binding protein preparation, was tyrosine-phosphorylated under the same conditions. Phosphorylation of both proteins was enhanced by human insulin. These results obtained by immunoprecipitation and immunoblotting using specific anti-phosphoserine/threonine/tyrosine antibodies were confirmed by phosphoamino acid analysis of the individual immunoprecipitated and gel-purified 32P-labeled phosphoproteins. During gel filtration, the 53-kDa protein coeluted as a 300-kDa complex together with the 70-kDa phosphotyrosine-containing protein and was coimmunoprecipitated with the latter using an anti-phosphotyrosine antibody, strongly arguing for complex formation between the two proteins. The data presented raise the possibility that stimulation of glycogen synthesis by insulin in yeast is mediated by a 53-kDa insulin-binding protein and a 70-kDa phosphotyrosine-containing protein which are organized in a large plasma membrane-bound signaling complex. Elucidation of the function and molecular mode of interaction of these components in yeast may help to understand metabolic insulin signaling in mammalian cells.

Insulin signaling in the yeast Saccharomyces cerevisiae. 2. Interaction of human insulin with a putative binding protein.

A putative insulin-binding protein (Kd = 0.5 +/- 0.2 microM for human insulin) was partially purified from solubilized plasma membranes of Saccharomyces cerevisiaeby wheat germ agglutinin and insulin affinity chromatographies. The binding affinities of various mutant insulin analogues correlated well with their capacities to activate glycogen synthase and SNF1 kinase in glucose-induced yeast spheroplasts, the ranking of their relative efficacies in yeast and in isolated rat adipocytes being similar. Using a bifunctional cross-linker and two different experimental protocols, a 53-kDa polypeptide contained in the insulin-binding protein preparation was specifically affinity cross-linked to [125I]monoiodo[B26]insulin. The relative rankings of the insulin analogues with respect to inhibition of cross-linking and binding to the partially purified insulin-binding protein were identical. Incubation of intact yeast spheroplasts with [125I]monoiodo[AI4]insulin led to specific and time-dependent association of the radiolabeled insulin with the cell surface followed by its internalization and degradation. These processes were considerably delayed by low temperature and energy depletion of the spheroplasts, suggesting involvement of the ATP-dependent endosomal apparatus. These data provide evidence for the existence of a low-affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae.

Inhibition of Insulin Receptor Activation by Insulin-like Growth Factor Binding Proteins

The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor beta subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3((1-87))), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.

Thyrotropin via Cyclic AMP Induces Insulin Receptor Expression and Insulin Co-stimulation of Growth and Amplifies Insulin and Insulin-like Growth Factor Signaling Pathways in Dog Thyroid Epithelial Cells

Despite the similarity of their receptors and signal transduction pathways, insulin is regarded as a regulator of glucose, protein, and lipid metabolism, whereas insulin-like growth factors (IGF-I and IGF-II) mainly act as mitogenic hormones. In the dog thyroid primary culture model, the triggering of DNA synthesis by thyrotropin (TSH) through cAMP, or by cAMP-independent factors including epidermal growth factor, hepatocyte growth factor and phorbol esters, requires insulin or IGFs as comitogenic factors. In the present study, in TSH-treated cells, IGF-I receptors and insulin receptors were paradoxically equivalent in their capacity to elicit the comitogenic pathway, which, however, was mediated only by IGF-I receptors in dog thyroid cells stimulated by cAMP-independent mitogens. Moreover, prior cell exposure to TSH or forskolin increased their responsiveness to insulin, IGF-I, and IGF-II, as seen on DNA synthesis and activation of a common insulin/IGF signaling pathway. To understand these observations, binding characteristics and expression of insulin and IGF-I receptors were examined. To analyze IGF-I receptor characteristics, the unexpected interference of a huge presence of IGF-binding proteins at the cell membrane was avoided using labeled Long R3 IGF-I instead of IGF-I. Strikingly, TSH, through cAMP, time-dependently induced insulin binding and insulin receptor mRNA and protein accumulation without any effect on IGF-I receptors. These findings constitute a first example of an induction of insulin receptor gene expression by a cAMP-mediated hormone. In dog thyroid cells, this allows low physiological insulin concentrations to act as a comitogenic factor and might explain in part the enhanced responsiveness to IGFs in response to TSH. This raises the possibility that TSH-insulin interactions may play a role in the regulation of thyroid growth and function in vivo.

Insulin-Induced Outgrowth of Pseudopodial Cables from Cultured Micromere-Derived Cells Isolated from Sea Urchin Embryos at the 16 Cell Stage, with Special Reference to the Insulin-Receptor.: (sea urchin/micromere/insulin/psedopodial cable/receptor).

Cultured cells derived from micromeres isolated from sea urchin embryos at the 16 cell stage are known to show outgrowth of pseudopodial cables followed by spicule rod formation when cultured in the presence of horse serum. Micromere-derived cells cultured with bovine insulin showed pseudopodial cable growth but did not produce spicule rods. Micromere-derived cells reversibly bound to insulin through out the period between 3 and 20 hr of culture. The dissociation constant of insulin with these cells was about 5.1 × 10-10 M during the whole culture period examined. Horse serum, as well as blastocoelic fluid obtained from early gastrulae, concentration-dependently reduced the amount of insulin bound to these cells, but the bound insulin was scarcely replaced by any proteins tested, such as bovine serum albumin. The micromere-derived cells were bound to have an insulin-binding protein, that may be the receptor for insulin or insulin-like proteins. The insulin-binding protein had a smaller molecular weight than the insulin receptor of mammalian cells. The binding of insulin with this protein in micromere-derived cells probably results in pseudopodial cable growth.

A new transgenic mouse model of chronic hyperglycemia.

Expression under the control of the mouse transferrin promoter of a transgene encoding a soluble secreted derivative of the ectodomain of the human insulin receptor in transgenic mice results in the accumulation of this high-affinity insulin-binding protein in the plasma. Alterations of glucose homeostasis are observed including postabsorptive hyperglycemia concomitant with increased hepatic glucose production and hyperinsulinemia. Thus, this is the first transgenic animal model of chronic hyperglycemia with alterations in glucose homeostasis that are produced without a targeted alteration of pancreatic function. These mice provide a new experimental model to follow the progression and long-term consequences of chronic hyperglycemia.