RNA interference against the putative insulin receptor substrate gene IRS1 affects growth and development in the pest natural enemy Pardosa pseudoannulata.

BACKGROUND: Insulin signalling pathways play crucial roles in regulating growth and development in insects, but their effects on the growth and development of Arachnids, such as spiders, have rarely been studied. As a valuable pest natural enemy in agricultural fields, the molecular mechanisms of insulin signalling pathway-mediated growth and development of the wolf spider, Pardosa pseudoannulata, are of particular interest. RESULTS: In this study, we identified and characterized six insulin signalling pathway genes - InR, InR2, IRS1, PI3K1, PI3K2, and PDK - in Pardosa pseudoannulata. Real-time quantitative polymerase chain reaction results were used to analyse the relative expression levels of the six genes in different developmental instars and tissues, and in response to starvation treatment. In addition, the function of the insulin receptor substrate (IRS1) gene was investigated using RNA interference technology, which found that IRS1 significantly influenced nutrient content, developmental duration, body weight, and gonad development. CONCLUSION: This study revealed the roles of six key insulin signalling pathway genes in Pardosa pseudoannulata, and in particular the importance of the IRS1 gene in regulating growth and development in the spider. The results lay the foundation for further research on the internal regulation mechanisms of growth and development in Araneae species, and also provide a reference for the artificial breeding of spiders. © 2023 Society of Chemical Industry.

Luteolin promotes pathogen resistance in Caenorhabditis elegans via DAF-2/DAF-16 insulin-like signaling pathway.

The DAF-2/DAF-16 insulin-like signaling pathway was an evolutionarily conserved pathway, which regulated many aspects of organismal physiology, such as pathogen resistance, metabolism, stress response, longevity. Luteolin, a flavone contained in many medical plants and in vegetables, had been shown to exhibit activities such as anti-tumor, anti-oxidant and neuroprotective effects. However, whether the Luteolin influenced the immune response and the underlying molecular mechanisms remained obscure. We found that Luteolin increased resistance to not only the Gram-negative pathogens Pseudomonas aeruginosa and Salmonella enterica but also the Gram-positive pathogens Enterococcus faecalis and Staphylococcus aureus in dose dependent manner. Meanwhile, Luteolin promoted host immune response via inhibiting the growth of pathogenic bacteria. Through the genetic screening in C. elegans, we found that Luteolin promoted innate immunity via DAF-2/DAF-16 insulin-like signaling pathway rather than p38 MAPK pathway and SKN-1. Furthermore, Luteolin activated the DAF-16/FOXO transcription factor for innate immune response. Our work suggested that Luteolin had the potential of improving the patients with pathogen infection.

The mammalian insulin antagonist S961 does not exhibit insulin receptor antagonism in rainbow trout in vivo.

Due to their reported 'glucose-intolerant' phenotype, rainbow trout have been the focus of comparative studies probing underlying endocrine mechanisms at the organismal, tissue and molecular level. A particular focus has been placed on the investigation of the comparative role of insulin, an important glucoregulatory hormone, and its interaction with macronutrients. A limiting factor in the comparative investigation of insulin is the current lack of reliable assays to quantify circulating mature and thus bioactive insulin. To circumvent this limitation, tissue-specific responsiveness to postprandial or exogenous insulin has been quantified at the level of post-translational modifications of cell signalling proteins. These studies revealed that the insulin responsiveness of these proteins and their post-translational modifications are evolutionarily highly conserved and thus provide useful and quantifiable proxy indices to investigate insulin function in rainbow trout. While the involvement of specific branches of the intracellular insulin signalling pathway (e.g., mTor) in rainbow trout glucoregulation have been successfully probed through pharmacological approaches, it would be useful to have a functionally validated insulin receptor antagonist to characterize the glucoregulatory role of the insulin receptor pathway in its entirety for this species. Here, we report two separate in vivo experiments to test the ability of the mammalian insulin receptor antagonist, S961, to efficiently block insulin signalling in liver and muscle in response to endogenously released insulin and to exogenously infused bovine insulin. We found that, irrespective of the experimental treatment or dose, activation of the insulin pathway in liver and muscle was not inhibited by S961, showing that its antagonistic effect does not extend to rainbow trout.

Behavioral and Transcriptional Effects of Short or Prolonged Fasting on the Memory Performances of Lymnaea stagnalis.

INTRODUCTION: The Garcia effect, a solid learning paradigm, was used to investigate the molecular and behavioral effects induced by different lengths of fasting on the cognitive functions in the pond snail Lymnaea stagnalis, a valid model system. METHODS: Three experimental groups were used: moderately hungry snails, food-deprived for 1 day (D1 snails), severely hungry snails (D5 snails), fasting for 5 days, and satiated snails with ad libitum access to food (AL snails). In the Garcia effect, a single pairing of an appetitive stimulus with a heat stressor results in a learned taste-specific negative hedonic shift. D5 snails were injected with bovine insulin and D1 snails with the insulin receptor antibody (Ab). As a control group, AL snails were injected with saline. Gene expression analyses were performed by real-time PCR in snails' central nervous system (CNS). RESULTS: AL snails are "average learners," D1 snails are the best performers, whereas the D5 ones do not show the Garcia effect. Severely fasting snails injected with insulin 3 h before the training procedure show the Garcia effect, whereas injecting 1-day fasting snails with insulin receptor Ab blocks their ability to express memory. The differences in memory performances are associated with changes in the expression levels of selected targets involved in neuronal plasticity, energy homeostasis, and stress response. DISCUSSION: Our results suggest that short-term fasting creates an optimal internal state in L. stagnalis' CNS, allowing a spike in insulin release and an upregulation of genes involved in neuroplasticity. Long-term fasting, instead, upregulates genes involved in energy homeostasis and animal survival.

A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity.

AIMS: Cardiotoxicity leading to heart failure (HF) is a growing problem in many cancer survivors. As specific treatment strategies are not available, RNA discovery pipelines were employed and a new and powerful circular RNA (circRNA)-based therapy was developed for the treatment of doxorubicin-induced HF. METHODS AND RESULTS: The circRNA sequencing was applied and the highly species-conserved circRNA insulin receptor (Circ-INSR) was identified, which participates in HF processes, including those provoked by cardiotoxic anti-cancer treatments. Chemotherapy-provoked cardiotoxicity leads to the down-regulation of Circ-INSR in rodents and patients, which mechanistically contributes to cardiomyocyte cell death, cardiac dysfunction, and mitochondrial damage. In contrast, Circ-INSR overexpression prevented doxorubicin-mediated cardiotoxicity in both rodent and human cardiomyocytes in vitro and in a mouse model of chronic doxorubicin cardiotoxicity. Breast cancer type 1 susceptibility protein (Brca1) was identified as a regulator of Circ-INSR expression. Detailed transcriptomic and proteomic analyses revealed that Circ-INSR regulates apoptotic and metabolic pathways in cardiomyocytes. Circ-INSR physically interacts with the single-stranded DNA-binding protein (SSBP1) mediating its cardioprotective effects under doxorubicin stress. Importantly, in vitro transcribed and circularized Circ-INSR mimics also protected against doxorubicin-induced cardiotoxicity. CONCLUSION: Circ-INSR is a highly conserved non-coding RNA which is down-regulated during cardiotoxicity and cardiac remodelling. Adeno-associated virus and circRNA mimics-based Circ-INSR overexpression prevent and reverse doxorubicin-mediated cardiomyocyte death and improve cardiac function. The results of this study highlight a novel and translationally important Circ-INSR-based therapeutic approach for doxorubicin-induced cardiac dysfunction.

Galanin enhanced insulin-mediated intracellular signaling by regulating the stability of membrane-localized insulin/IR.

Previous studies have shown that insulin has the important regulatory effect on the intestinal tract. However, until now, the biological properties of insulin on intestinal cell has not been revealed. Therefore, in the current research, we first studied the cell characteristics and signaling profiles of insulin in the intestinal cell model, and found that insulin can be internalized into the cytoplasm in a time-dependent manner. After internalization, insulin transported into different type of endosomes. More importantly, we explored the effect of galanin on insulin-mediated signaling pathways (galanin is a polypeptide composed of 29 amino acid residues, galanin is widely distributed in the central and peripheral nervous system and has a variety of biological activities), and found that galanin can increase insulin sensitivity by regulating insulin receptor (IR)-mediated signal transduction pathways. We further study the potential molecular mechanism by which galanin enhances insulin sensitivity, and found that galanin could increase the time of insulin acting on the cell membrane. Further experiments showed that galanin could stabilize the membrane-localized insulin/IR, which may be an important new potential mechanism by which galanin improves the biological activity of insulin. This study laid the foundation for exploring the relationship between galanin and insulin sensitivity.

The impact of HCV eradication on hyperglycemia, insulin resistance, cytokine production, and insulin receptor substrate-1 and 2 expression in patients with HCV infection.

Virological responses after hepatitis C virus (HCV) treatment may alleviate liver disease and extra-hepatic manifestations. Our study aims to explore the impact of HCV eradication on the glycemic status, insulin resistance, cytokine production, and insulin receptor substrate (IRS)-1 and 2 gene expression levels in HCV-hyperglycemic patients. A total of 90 participants were allocated as follows: Group 1 included 30 healthy subjects as controls, and Group 2 included 60 HCV-hyperglycemic patients treated with a direct-acting antiviral (DAA) regimen and further subdivided into HCV-pre-diabetic and HCV-diabetic groups. Laboratory assays screened patients before and after treatments. Our data showed an excellent rate of virological responses in HCV groups after HCV treatment. Moreover, HCV eradication significantly ameliorated blood glucose levels and insulin resistance biomarkers in HCV-hyperglycemic patients compared with baseline values. Also, interleukin (IL)-6, IL-17, IL-23, and IL-27 levels were significantly ameliorated after viral clearance in HCV-hyperglycemic patients compared with baseline values. Similarly, IRS-1 and 2 mRNA expression levels were upregulated in these patients post-HCV treatment compared with baseline values. HCV clearance ameliorated hyperglycemia, cytokine production, and enhanced insulin sensitivity. Future researches will be needed to explore the effects of cytokines and IRS on HCV infection and treatment on a large cohort.

Estrogen receptor variant ERα46 and insulin receptor drive in primary breast cancer cells growth effects and interleukin 11 induction prompting the motility of cancer-associated fibroblasts.

Among the prognostic and predictive biomarkers of breast cancer (BC), the role of estrogen receptor (ER)α wild-type has been acknowledged, although the action of certain ERα splice variants has not been elucidated. Insulin/insulin receptor (IR) axis has also been involved in the progression and metastasis of BC. For instance, hyperinsulinemia, which is often associated with obesity and type 2 diabetes, may be a risk factor for BC. Similarly, an aberrant expression of IR or its hyperactivation may correlate with aggressive BC phenotypes. In the present study, we have shown that a novel naturally immortalized BC cell line (named BCAHC-1) is characterized by a unique expression of 46 kDa ERα splice variant (ERα46) along with IR. Moreover, we have shown that a multifaceted crosstalk between ERα46 and IR occurs in BCAHC-1 cells upon estrogen and insulin exposure for growth and pulmonary metastasis. Through high-throughput RNA sequencing analysis, we have also found that the cytokine interleukin-11 (IL11) is the main factor linking BCAHC-1 cells to breast cancer-associated fibroblasts (CAFs). In particular, we have found that IL11 induced by estrogens and insulin in BCAHC-1 cells regulates pro-tumorigenic genes of the "extracellular matrix organization" signaling pathway, such as ICAM-1 and ITGA5, and promotes both migratory and invasive features in breast CAFs. Overall, our results may open a new scientific avenue to identify additional prognostic and therapeutic targets in BC.

Heparin impairs skeletal muscle glucose uptake by inhibiting insulin binding to insulin receptor.

AIM: Heparin, a widely used antithrombotic drug has many other anticoagulant-independent physiological functions. Here, we elucidate a novel role of heparin in glucose homeostasis, suggesting an approach for developing heparin-targeted therapies for diabetes. METHODS: For serum heparin levels and correlation analysis, 122 volunteer's plasma, DIO (4 weeks HFD) and db/db mice serums were collected and used for spectrophotometric determination. OGTT, ITT, 2-NBDG uptake and muscle GLUT4 immunofluorescence were detected in chronic intraperitoneal injection of heparin or heparinase (16 days) and muscle-specific loss-of-function mice. In 293T cells, the binding of insulin to its receptor was detected by fluorescence resonance energy transfer (FRET), Myc-GLUT4-mCherry plasmid was used in GLUT4 translocation. In vitro, C2C12 cells as mouse myoblast cells were further verified the effects of heparin on glucose homeostasis through 2-NBDG uptake, Western blot and co-immunoprecipitation. RESULTS: Serum concentrations of heparin are positively associated with blood glucose levels in humans and are significantly increased in diet-induced and db/db obesity mouse models. Consistently, a chronic intraperitoneal injection of heparin results in hyperglycaemia, glucose intolerance and insulin resistance. These effects are independent of heparin's anticoagulant function and associated with decreases in glucose uptake and translocation of glucose transporter type 4 (GLUT4) in skeletal muscle. By using a muscle-specific loss-of-function mouse model, we further demonstrated that muscle GLUT4 is required for the detrimental effects of heparin on glucose homeostasis. CONCLUSIONS: Heparin reduced insulin binding to its receptor by interacting with insulin and inhibited insulin-mediated activation of the PI3K/Akt signalling pathway in skeletal muscle, which leads to impaired glucose uptake and hyperglycaemia.

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells.

Wolbachia-infected mosquitoes are refractory to super-infection with arthropod-borne pathogens, but the role of host cell signaling proteins in pathogen-blocking mechanisms remains to be elucidated. Here, we use an antibody microarray approach to provide a comprehensive picture of the signaling response of Aedes aegypti-derived cells to Wolbachia. This approach identifies the host cell insulin receptor as being downregulated by the bacterium. Furthermore, siRNA-mediated knockdown and treatment with a small-molecule inhibitor of the insulin receptor kinase concur to assign a crucial role for this enzyme in the replication of dengue and Zika viruses in cultured mosquito cells. Finally, we show that the production of Zika virus in Wolbachia-free live mosquitoes is impaired by treatment with the selective inhibitor mimicking Wolbachia infection. This study identifies Wolbachia-mediated downregulation of insulin receptor kinase activity as a mechanism contributing to the blocking of super-infection by arboviruses.

A novel recombinant peptide INSR-IgG4Fc (Yiminsu) restores insulin sensitivity in experimental insulin resistance models.

Type 2 diabetes mellitus (T2DM) is a chronic degenerative endocrine and metabolic disease with high mortality and morbidity, yet lacks effective therapeutics. We recently generated a novel fusion peptide INSR-IgG4Fc, Yiminsu (YMS), to facilitate the high-affinity binding and transportation of insulin. Thus, the aim of the present study was to determine whether the novel recombinant peptide, YMS, could contribute to restoring insulin sensitivity and glycaemic control in insulin resistance models and revealing its underlying mechanism. Palmitic acid (PA)-treated LO2 cells and high fat diet (HFD)-fed mice were treated with YMS. Therapeutic effects of YMS were measured using Western blotting, ELISA, qPCR, Histology and transmission electron microscopy. We observed that YMS treatment effectively improved insulin signaling in PA-treated LO2 cells and HFD-fed mice. Notably, YMS could significantly reduce serum levels of glucose, triglycerides, fatty acids and cholesterol without affecting the serum insulin levels. Moreover, our data demonstrated that YMS could restore glucose and lipid homeostasis via facilitating insulin transportation and reactivating PI3K/Akt signaling in both PA-treated cells and liver, gastrocnemius and brown fat of HFD-fed mice. Additionally, we noticed that the therapeutic effects of YMS was similar as rosiglitazone, a well-recognized insulin sensitizer. Our findings suggested that YMS is a potentially candidate for pharmacotherapy for metabolic disorders associated with insulin resistance, particularly in T2DM.

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic ß-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity.

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP7, is at a high level in pancreatic ß-cells and is important for insulin exocytosis. To better understand IP7 regulation in ß-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP7. Although the inhibitors have diverse targets, they all perturbed IP7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high Km for ATP makes IP7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic ß-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP7's role in cellular signal transduction is likely to have been underestimated.

Autocrine regulation of insulin secretion.

Impaired insulin secretion from pancreatic ß-cells is a major factor in the pathogenesis of type 2 diabetes. The main regulator of insulin secretion is the plasma glucose concentration. Insulin secretion is modified by other nutrients, circulating hormones and the autonomic nervous system, as well as local paracrine and autocrine signals. Autocrine signalling involves diffusible molecules that bind to receptors on the same cell from which they have been released. The first transmitter to be implicated in the autocrine regulation of ß-cell function was insulin itself. The importance of autocrine insulin signalling is underscored by the finding that mice lacking insulin receptors in ß-cells are glucose intolerant. In addition to insulin, ß-cells secrete a variety of additional substances, including peptides (e.g. amylin, chromogranin A and B and their cleavage products), neurotransmitters (ATP and γ-aminobutyric acid) and ions (e.g. zinc). Here we review the autocrine effects of substances secreted from ß-cells, with a focus on acute effects in stimulus-secretion coupling, present some novel data and discuss the general significance of autocrine signals for the regulation of insulin secretion.

Modulation of ß-cell function: a translational journey from the bench to the bedside.

Both decreased insulin secretion and action contribute to the pathogenesis of type 2 diabetes (T2D) in humans. The insulin receptor and insulin signalling proteins are present in the rodent and human ß-cell and modulate cell growth and function. Insulin receptors and insulin signalling proteins in ß-cells are critical for compensatory islet growth in response to insulin resistance. Rodents with tissue-specific knockout of the insulin receptor in the ß-cell (ßIRKO) show reduced first-phase glucose-stimulated insulin secretion (GSIS) and with aging develop glucose intolerance and diabetes, phenotypically similar to the process seen in human T2D. Expression of multiple insulin signalling proteins is reduced in islets of patients with T2D. Insulin potentiates GSIS in isolated human ß-cells. Recent studies in humans in vivo show that pre-exposure to insulin increases GSIS, and this effect is diminished in persons with insulin resistance or T2D. ß-Cell function correlates to whole-body insulin sensitivity. Together, these findings suggest that pancreatic ß-cell dysfunction could be caused by a defect in insulin signalling within ß-cell, and ß-cell insulin resistance may lead to a loss of ß-cell function and/or mass, contributing to the pathophysiology of T2D.

Resistencia a insulina en el músculo esquelético: conexión con la obesidad / Obesity-Associated Insulin resistance in skeletal muscle

Entre las complicaciones asociadas a la Obesidad, tiene una especial relevancia el desarrollo de resistencia a la insulina, siendo el primer eslabón de una amplia patología conocida como diabetes tipo 2. La Obesidad se considera como un estado crónico de inflamación de baja intensidad, como indican los niveles circulantes elevados de moléculas proinflamatorias. Se ha propuesto al TNFα como el nexo de unión entre adiposidad y desarrollo de resistencia a insulina ya que la mayoría de los pacientes con diabetes tipo 2 son obesos y tienen aumentada la expresión de TNFα en sus adipocitos, y los animales obesos deleccionados para la función del TNFα o su receptor no desarrollan resistencia a insulina. Las citocinas proinflamatorias producidas por los adipocitos y/o macrófagos activan quinasas de estrés, proinflamatorias y factores de transcripción que actúan sobre los tejidos periféricos (entre ellos el músculo y el propio tejido adiposo) produciendo resistencia a la acción de la insulina, que es un defecto en la señalización a varios niveles. En concreto, el TNFα activa la quinasa p38MAPK que fosforila en residuos de serina a los IRSs, bloqueando su fosforilación en tirosina en respuesta a la insulina, tanto en adipocitos marrones como en miocitos. Muy recientemente hemos observado que la fosfatasa PTP1B también está implicada en la resistencia a insulina por TNFα en ambos modelos. En la clínica se está utilizando actualmente el tratamiento con tiazolidindionas en pacientes con diabetes tipo 2. Otros agonistas de receptores nucleares empiezan a aparecer en la bibliografía como potenciales sensibilizadores a acción de la insulina, entre ellos el LXR, que puede antagonizar la señalización proinflamatoria en los propios adipocitos y/o en el músculo (AU)

Insulin resistance is an important contributor to the pathogenesis of type 2 diabetes and obesity is a risk factor for its development, due in part to the fact that adipose tissue secretes proteins called adipokines that may influence insulin sensitivity. Among these molecules, TNFα has been proposed as a link between obesity and insulin resistance because TNFα is overexpressed in adipose tissues of obese animals and humans, and obese mice lacking either TNFα or its receptor show protection for developing insulin resistance. The direct exposure to TNFα induced a state of insulin resistance on glucose uptake in myocytes and brown adipocytes, due to the activation of pro-inflammatory pathways that impair insulin-signaling at the level of the IRS proteins. In this regard the residue Ser307 in IRS-1 has been identified as a site for TNFα- inhibitory effects in myotubes, with being p38MAPK and IKK involved in the phosphorylation of this residue. Conversely, serine phosphorylation of IRS-2 mediated by TNFα activation of MAPKs was the mechanism found in brown adipocytes. The phosphatase PTP1B acts as a physiological negative regulator of insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor and IRS-1, and PTP1B expression is increased in muscle and white adipose tissue of obese and diabetic humans and rodents. Moreover, up-regulation of PTP1B expression has recently been found in cells treated with TNFα. Accordingly, myocytes and primary brown adipocytes deficient on PTP1B are protected against insulin resistance by this cytokine. Furthermore, down-regulation of PTP1B activity is also possible by the use of pharmacological agonists of nuclear receptors that restored insulin sensitivity in the presence of TNFα. In conclusion, the lack of PTP1B in muscle and brown adipocytes increase insulin sensitivity and glucose uptake and could confer protection against insulin resistance induced by adipokines (AU)

Lipogenesis stimulatory and inhibitory activities of the insulin mediators.

Incubating plasma membranes prepared from pig liver with varying concentrations of insulin (50-1000 microU/ml) resulted in the release of at least two insulin chemical mediators. One of them was fraction 1 of insulin mediator (M. W. 3700-4000 daltons) which had a significant lipogenesis-stimulating activity. The other was fraction 2 of insulin mediator (M. W. about 1000 daltons) which exhibited a lipogenesis-inhibitory activity. The ratio of yield between the two mediators produced from the membranes was not only dependent on the concentration but also on the potency of insulin and its analogs added. The result showed that there was more production of fraction 2 than fraction 1 with the inducer at low concentration (100 microU/ml), while the production of fraction 1 from the plasma membranes incubated with high concentration of insulin (300 microU/ml) was higher than fraction 2. On the other hand, insulin and its analogs which have different biological activities and receptor binding activities have been used to induce the insulin mediators. The results obtained were similar to those mentioned above. This suggested that the generation of the mediators was dependent on the biological potences but not the binding activities.

Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor.

Recently, we have described a COOH-terminal deletion mutation of the human insulin receptor (HIR delta CT) that exhibits normal insulin-mediated kinase activity and endocytosis, but is inefficient in stimulating glucose transport and glycogen synthase (McClain, D. A., Maegawa, H., Levy, J., Huecksteadt, T., Dull, T. J., Lee, J., Ullrich, A., and Olefsky, J.M. (1988) J. Biol. Chem. 263, 8904-8911; Maegawa, H., McClain, D. A., Freidenberg, G., Olefsky, J. M., Napier, M., Lipari, T., Dull, T. J., Lee, J., and Ullrich, A. (1988) J. Biol. Chem. 263, 8912-8917). In this paper, we report that despite this defect in metabolic signaling, the truncated receptor exhibits augmented mitogenic activity compared to normal receptors. These results were verified in three independently isolated clones of Rat 1 fibroblasts transfected with the HIR delta CT cDNA. The increase in insulin sensitivity of mitogenic stimulation was proportional to the number of HIR delta CT receptors expressed on the cells. By contrast, only the cells with normal receptors and none of the HIR delta CT clones exhibit increased sensitivity for a metabolic action of insulin, the stimulation of glucose uptake. Stimulation of cells by other mitogens and autoradiographic analysis confirm that the enhanced mitogenic effects seen in HIR delta CT cells are attributable only to the presence of the truncated insulin receptors. These receptors mediate the tyrosine phosphorylation of a number of cellular proteins, and the pattern of these phosphorylations differs quantitatively from that seen in cells with normal receptors. We conclude: 1) The COOH terminus plays a role in signaling metabolic actions of insulin, perhaps through its recognition of substrates for the receptor kinase. 2) By contrast, the COOH terminus is an inhibitory regulator of mitogenesis, and removal of the terminal 43 amino acids converts the receptor from a moderately active growth signaler to a very active one. 3) The changes seen in biologic activities of the HIR delta CT receptor are associated with quantitative changes in substrate phosphorylation by the receptor kinase.

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density.

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.

A putative mediator of insulin action which inhibits adenylate cyclase and adenosine 3',5'-monophosphate-dependent protein kinase: partial purification from rat liver: site and kinetic mechanism of action.

A novel putative mediator of insulin action which acts to inhibit adenylate cyclase and cAMP-dependent protein kinase has been purified from livers of insulin-treated streptozotocin-diabetic rats. It was increased by short term (5-min) insulin injections in vivo and purified several thousand-fold by Sephadex and HPLC. Its mol wt was somewhat larger (2500) than previous mediators identified, and it was more hydrophobic in character. Its mechanism of action or adenylate cyclase was determined and found to be chiefly directed against the catalytic subunit. Its action on the cAMP-dependent protein kinase was found to be competitive with regard to protein substrate, but noncompetitive with regard to ATP and cAMP. Its relationship to other putative insulin mediators and the mechanism of insulin action is discussed.

Stimulation of pyruvate dehydrogenase activity in intact rat adipocytes by insulin mediator from rat skeletal muscle.

This study compared the effects of insulin and insulin mediator from skeletal muscle of control and insulin-treated rats on intact adipocyte pyruvate dehydrogenase. Increasing insulin concentrations stimulated pyruvate dehydrogenase activity in a biphasic manner with a maximal stimulation at 100 microU/ml which was 2-fold and sustained for up to 1 h. The mediators from control or insulin-treated rats also stimulated pyruvate dehydrogenase of intact adipocytes with the effect increasing in a linear manner up to a 1:10 final dilution. The latter mediator had twice the stimulatory activity as the former. Peak stimulation of pyruvate dehydrogenase by the mediators was attained within 10 min of incubation. The enzyme activity rapidly declined thereafter, with the stimulation by mediator from control rats decreasing at a faster rate than that due to mediator from insulin-treated rats. The stimulatory effect of the mediators on adipocyte pyruvate dehydrogenase was found to be additive to that of insulin. This study demonstrates: 1) that insulin mediator can act on mitochondrial pyruvate dehydrogenase of intact, functional adipocytes as it does on isolated intact or broken mitochondria; 2) that the mediator is degraded by the adipocyte; and 3) that the amount of mediator generated by insulin probably limits the stimulation of pyruvate dehydrogenase by insulin. These findings further substantiate the physiological relevance of this putative insulin second messenger.