Increase In Insulin Binding Research Articles

Regulation of insulin entry into the brain

AbstractBackgroundCentral nervous system (CNS) insulin has repeatedly been shown to play a role in cognition. Little, if any, insulin is synthesized in the brain and therefore requires navigation of the blood‐brain barrier (BBB) to act. The BBB is a unique structure in the brain that regulates the entry of serum substrates and can act as both a barrier and signaling structure. We hypothesize the BBB is a critical structure involved in the regulation of CNS insulin signaling and CNS insulin availability. We have investigated the regulation of insulin BBB transport and interactions under various conditions including exercise, brain insulin resistance, and have begun to explore the mechanisms for insulin transport across the BBB.MethodFor exercise, male and female mice were habituated to locked voluntary running wheels. After habituation, wheels were unlocked in some cages (exercise) for 24 hours while remaining locked (sedentary) in the others. For brain insulin resistance, mice were treated with an intracerebroventricular delivery of the insulin receptor antagonist, S961. For mechanisms involved in insulin BBB transport and interactions, endocytosis inhibitors were used to differentiate between clathrin and caveolin endocytosis. Following manipulation, insulin BBB pharmacokinetics were assessed with the quantitative multiple‐time regression analysis using radioactively labeled insulin and albumin, a marker for vascular space. Mice were injected intravenously or perfused in situ with the radiolabeled substrates and at timepoints up to 10 min, blood and brains collected and radioactivity measured. 125I‐insulin entry and binding to the BBB was assessed.ResultExercise enhanced 125I‐insulin interactions at the BBB. Specifically, exercise increased insulin BBB transport by two‐fold in males and increased insulin binding at the BBB in females. Brain insulin resistance impaired 125I‐insulin transport across the BBB, particularly in females. Lastly, caveolin‐mediated endocytosis may be involved in hypothalamic 125I‐insulin BBB transport.ConclusionWe have shown insulin BBB transport is highly regulated and can be impacted by exercise, brain insulin resistance, and may involve various endocytic pathways in a regional‐dependent manner. Continued investigation on the pathways involved in insulin BBB transport is warranted to aid in treating or preventing brain insulin resistance as occurs in Alzheimer’s disease.

New insights into serum/extracellular thioredoxin in regulating hepatic insulin receptor activation

BackgroundSerum thioredoxin of type-2 diabetic patients is significantly higher than that of healthy people. Pathophysiological significance is unclear. MethodsEffects of serum/extracellular thioredoxin on phosphorylation (activation) of hepatic insulin receptor (IR) were investigated by using methods in biochemistry, cell/molecular biology and mass spectrometry. ResultsIn human serum, thioredoxin and insulin may interact. Their mixture contains a mixed disulfide between insulin B-chain and thioredoxin-Cys73, which limits their activities. In contrast, free form of serum/extracellular thioredoxin is active, and can regulate phosphorylation of insulin receptor β-subunits (IRβ) via direct/indirect mechanisms. The direct mechanism associates with positive regulation. Serum/extracellular thioredoxin increases insulin binding to IR, facilitating insulin-induced phosphorylation of IRβ and downstream AKT. The indirect mechanism is involved in negative regulation. Entry of extracellular thioredoxin into hepatic cells via IR enhances the expression and activity of cellular protein-tyrosine phosphatase 1B (PTP1B), which negatively regulates IRβ phosphorylation. After coordination between these two mechanisms, the positive impact of serum/extracellular thioredoxin overwhelms its negative impact on IRβ phosphorylation, which subsequently accelerates hepatic glucose uptake. In hepatic cells with thioredoxin deficiency, insulin-induced IRβ phosphorylation is decreased, which could be restored by extracellular thioredoxin entry. Moreover, the results from assaying 475 serum samples demonstrate a discriminating value of serum thioredoxin activity in diagnosing type-2 diabetes. ConclusionSerum/extracellular thioredoxin plays a critical role in regulating hepatic IRβ phosphorylation. General significanceIn case of insulin resistance/type-2 diabetes, hepatic IRβ is at low phosphorylation level, thereby the improvement effect of serum/extracellular thioredoxin on insulin-induced IRβ phosphorylation seems particularly important.

Sensitizing Aptamer INSR‐A43 Enhances Insulin Sensitivity and Sensitizes Insulin Receptor Signaling Pathways

Aptamers are single‐stranded oligonucleotides (DNA or RNA) and form a unique three‐dimensional (3D) structure based on its sequence. These aptamers have high affinity and specificity to target molecule and recognize 3D structure of target molecule surface. For this reason, aptamers have been in the spotlight as a universal capture. Especially, aptamers can modulate target protein as allosteric agonist or allosteric antagonist. However, aptamer‐based allosteric enhancer or sensitizer (hereafter ‐ sensitizer) has not been elucidated. Here, for the first time, we show the INSR‐A43, which is aptamer‐based sensitizer for the insulin receptor. Because insulin receptor plays a key role in regulation of glucose metabolism, it is important to specifically modulate the insulin receptor. INSR‐A43 binds to allosteric site of the insulin receptor and then increases insulin binding to the insulin receptor. Taken together, INSR‐A43 effectively enhances insulin sensitivity to the insulin receptor and sensitizes insulin receptor and insulin receptor‐related downstream signaling pathways. We expect that aptamers can be applied as an attractive tool for receptor sensitization.Support or Funding InformationThis research was supported by Grobal Research Laboratory (GRL) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. NRF‐2016K1A1A2912722)

Anti-Human Herpesvirus 8 antibodies affect both insulin and glucose uptake by virus-infected human endothelial cells

Human Herpesvirus 8 (HHV8) is known to be the cause of the malignant tumour named Kaposi's sarcoma. It is believed to induce an intense modification of cell metabolism in endothelial cells. In this work we analysed the role of anti-HHV8 antibodies in both the insulin and glucose uptake of HHV8-infected primary human endothelial cells (HUVEC). Western blotting, immunofluorescence and radiolabelled glucose were employed to assess the pPI3K expression, insulin binding and glucose-uptake by HUVEC cells, respectively. We confirmed that HHV8-infection is able to enhance both insulin binding and glucose-uptake in HHV8-infected primary endothelial cells; in addition, we found that anti-HHV8 specific antibodies are able to further increase both insulin and glucose uptake during the late latent phase of HHV8-infection in vitro. These findings suggest that a specific immune response to HHV8-infection may cooperate in boosting the cell metabolism, further enhancing the already increased insulin binding and glucose-uptake in HHV8-infected cells, which is a peculiar property of several oncogenic viruses.

Down-regulation of Type I Insulin-like Growth Factor Receptor Increases Sensitivity of Breast Cancer Cells to Insulin

The type I insulin-like growth factor receptor (IGF1R) and insulin receptor (IR) are structurally and functionally related heterotetrameric receptors. Activation of IGF1R has been shown to regulate breast cancer cell biology, and it has become an attractive therapeutic target. Most strategies have focused on targeting IGF1R alone without affecting IR levels given the known physiologic functions of IR. Human breast cancer cell lines and tissues revealed mRNA expression of both IGF1R and IR. Because alphabeta chains of IGF1R and IR form hybrid receptors, we hypothesized that agents solely targeting IGF1R may affect tumor biology mediated by IGF1R/IR hybrids and IR. We used small interfering RNA (siRNA) technology to specifically down-regulate IGF1R by 60% to 80% in the MDA-435/LCC6 cell line, which was sufficient to diminish activation of IGF1R by IGF-I. IGF1R down-regulation by siRNA did not affect IR levels but, interestingly, sensitized cells to insulin activation of downstream signaling pathways in several breast cancer cell lines. IGF1R siRNA treatment diminished hybrid receptor formation, suggesting that specific down-regulation of IGF1R resulted in enhanced holo-IR formation. In addition, IGF1R down-regulation increased insulin binding consistent with the formation of an increased number of holo-IR on the cell surface. Accordingly, insulin-stimulated glucose uptake was enhanced on IGF1R down-regulation. In conclusion, our data suggest that specific siRNA targeting of IGF1R alone in breast cancer increases insulin sensitivity. Because IR also activates signaling pathways similar to IGF1R in breast cancer cells, agents targeting both receptors may be necessary to disrupt the malignant phenotype regulated by this growth factor system.

The effect of annatto on insulin binding properties in the dog

Various plants are used in Caribbean folklore for the treatment of a variety of illnesses including diabetes mellitus. Preliminary investigations of several crude plant extracts have indicated that the annatto (Bixa orellana), among others, does in fact exhibit hypoglycaemic properties. This present investigation sought to isolate the hypoglycaemic principle(s) from the crude extract and to determine the mechanism of action. Purification experiments employing thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) resulted in an oil-soluble, partially purified annatto extract. The latter caused a decrease in blood glucose level of 5.62+/-0.13 (n=34) mmol/dL versus 6.31+/-0.12 (n=34) for the control (p<0.01) 1 h after administration. This hypoglycaemia persisted for an additional hour when the oral glucose tolerance test (OGTT) was performed on dogs treated with annatto and compared with the control. Plasma insulin levels measured at 1.0 h showed that there was an increase in plasma insulin levels of 59.57+/-8.3 microIU/mL for the annatto treated dogs versus 40.95+/-5.46 microIU/mL for the control (p<0.05), this elevation persisted throughout the duration of the OGTT which followed. Insulin receptor studies, using a modification of the method of Gambhir et al. done on mononuclear leucocytes and erythrocytes obtained from blood taken 1 h after administration showed that there was an increase in the percentage receptor binding when compared with the control. Insulin affinity results showed that there was an increase of 1.8+/-0.2x10(8) m-1 (n=12) in mononuclear leucocytes for the annatto treated dogs versus 1.2+/-0.2x10(8) m-1 for the control (p<0.05). In the enythrocytes, there was also an increase in affinity from 1.2+/-0.2x10(8) m-1 to 2.3+/-0.2x10(8) m-1 for the control and treated animals, respectively. In conclusion, it can be stated that annatto is responsible for the hypoglycaemic episodes seen in the dogs which was mediated by an increase in plasma insulin concentration as well as an increase in insulin binding on the insulin receptor due to elevated affinity of the ligand for the receptor.

Intrauterine hyperglycemia increases insulin binding sites but not glucose transporter expression in discrete brain areas in term rat fetuses.

Diabetic pregnancy results in several metabolic and hormonal disorders, both in the embryo and the fetus of different species, including humans. Insulin is a potent modulator of brain development and is suggested to promote the differentiation and maturation of hypothalamic or related extrahypothalamic structures, which are directly involved in neural inputs to the pancreas. Because these structures are known to be specifically responsive both to insulin and glucose, we examined the effects of 48-h hyperglycemic clamps in unrestrained pregnant rats on insulin binding and glucose transporter expression in hypothalamic and extrahypothalamic-related areas of their fetal offspring. The main result was an increase in insulin binding in the ventromedial hypothalamic nucleus (VMH), the arcuate nucleus (AN), and the lateral hypothalamus (LH), and in the nucleus of the tractus solitarius (NTS) for extrahypothalamic areas (+30% in the VMH, +37% in the AN, +25.8% in the LH, and +37.3% in the NTS). The deleterious effect of brain hyperinsulinism during the late gestational stage does not seem to act through glucose transporter (GLUT) expression, inasmuch as no relationship between GLUT level and hyperinsulinism in brain areas could be observed. The specific increase in insulin binding in areas involved in the nervous control of metabolism could be a factor in the increased glucose intolerance and impairment of insulin secretion that was previously observed in the adult rats from hyperglycemic mothers.

Beneficial Effect of Short-Term Endurance Training on Glucose Metabolism During Rehabilitation After Coronary Bypass Surgery

Dylewicz, P; Bienkowska, S; Szczesniak, L; Rychlewski, T; Przywarska, I; Wilk, M; Jastrzebski, AEditor(s): Cannistra, Lauralyn MD; Carlin, Brian MD; Keteyian, Steven J. PhD

Beneficial Effect of Short-term Endurance Training on Glucose Metabolism During Rehabilitation After Coronary Bypass Surgery

Our study was aimed at determining whether beneficial modification of carbohydrate metabolism can be obtained after a short-term training program and whether it is associated with an increase in binding and degradation of (125)I-insulin by erythrocyte receptors that suggests a decrease in insulin resistance. The study was conducted in a group of 20 patients aged 56 +/- 1.9 years (mean +/- SEM), within 1 to 6 months after coronary bypass surgery. All patients completed 15 training sessions based on 30 min of cycling with a constant load. Before and after a 3-week training program, glucose, insulin, and C-peptide blood levels, as well as binding and degradation of (125)I-insulin by erythrocyte receptors, were determined. A statistically significant decrease was found in the blood glucose level, from 111.2 +/- 4.2 to 97.8 +/- 3.5 mg/dL (p < 0.01); this decrease was not accompanied by significant insulin concentration changes. There was also a significant increase in insulin binding, from 0.535 +/- 0.059 to 0.668 +/- 0.042 pg (125)I/10(11) RBCs (p < 0.01), and degradation from 7.64 +/- 0.54 to 9.49 +/- 0.58 pg (125)I/10(11) RBCs (p < 0.05). The results indicated that even short-term endurance training in patients rehabilitated after coronary bypass surgery induced favorable modification of glucose metabolism, presumably caused by a decrease in insulin resistance.

Insulin receptor expression in primary and cultured osteoclast-like cells

Skeletal growth is the net product of coordinated bone formation and resorption. Insulin is known to stimulate bone formation by actions on osteoblasts. It is not known whether insulin receptors are present on osteoclasts, or whether insulin regulates osteoclastic function. We present here immunocytochemical evidence of insulin receptor expression by mature mono- and multinucleated murine osteoclast-like cells generated in vitro, and in primary neonatal rat and mouse osteoclasts. Radiolabeled studies indicated that progressive enrichment of osteoclast-like cells in coculture was associated with increased insulin binding. When osteoclast-like cells generated in vitro were plated onto dentine slices, insulin dose-dependently inhibited pit formation by up to 80%, suggesting a role for insulin in osteoclast function. These data are consistent with an effect of insulin on bone resorption in addition to those previously recognized on bone formation, actions that together result in net bone growth.

Expression and function of insulin/insulin-like growth factor I hybrid receptors during differentiation of 3T3-L1 preadipocytes.

During the assembly of cell surface receptors, insulin proreceptors are sometimes joined to insulin-like growth factor (IGF) receptor precursors to form covalently linked hybrid receptors. To address the biological consequences of hybrid receptor formation, we studied 3T3-L1 cells known to undergo a 50-70-fold increase in insulin binding while maintaining nearly constant levels of IGF-I binding during differentiation from preadipocytes into adipocytes. The presence of insulin/IGF receptor hybrids in 3T3-L1 adipocytes was demonstrated by the immunoprecipitation of phosphorylated receptors and a novel enzyme-linked immunoassay. Hybrid receptor levels were very low in the early stages of differentiation and increased rapidly between days 4 and 6, reaching a level about 100-fold higher in the mature adipocyte. Coincident with the hybrid assembly, the formation of archetypal (alpha2,beta2) IGF receptors decreased. In fully differentiated adipocytes, virtually all of the IGF receptors were in hybrid form. Stimulation by IGF-I of receptors isolated from mature adipocytes caused autophosphorylation of IGF receptor beta subunits in hybrid complexes, whereas autophosphorylated IGF holoreceptors were not demonstrable. Insulin and IGF-I were equipotent in stimulating glucose uptake in the differentiated adipocytes, leading to the conclusion that hybrid insulin/IGF receptors can transduce a transmembrane signal when activated by IGF-I. We conclude that hybrid formation constitutes a novel post-translational mechanism whereby increased synthesis of insulin receptors limits the cell surface expression of the homologous IGF receptor. Furthermore, biological actions in 3T3-L1 adipocytes, previously attributed to archetypal IGF receptors, are in fact mediated through hybrid receptors.

Effect of Long Lasting Terguride Treatment on Mutual Relationship Between Glycide and Lipid Parameters in SHR/N-cp Koletsky Rats

Experiments were performed in the genetically hypertensive obese rats of Koletsky type (SHR/N-cp) and in their lean siblings. The effect of long lasting terguride treatment on glycide and lipid metabolism was monitored. Terguride decreases insulinemia in all groups of rats. In all groups of rats terguride increases tolerance glucose. Terguride increases insulin binding to erythrocytes in all groups of rats except SHR/N-cp obese females. The mentioned drug decreases plasma triglycerides in SHR/N-cp obese females. On the other hand, this drug increases plasma triglycerides in SHR/N-cp obese males. Correlation between basal glycemia and insulin binding to erythrocytes as well as between triglycerides and insulinemia which was found in control SHR/N-cp lean males is missing under the terguride treatment. Similarly, correlation between plasma triglycerides and insulinemia, glucose tolerance and insulinemia, basal glycemia and insulinemia, plasma triglycerides and basal glycemia are missing under the terguride treatment in SHR/N-cp lean females. Under the terguride treatment there appears correlation between insulin binding to erythrocytes and basal glycemia. We found in SHR/N-cp obese males opposite changes in number of correlations when we compare control and terguride animals. While in controls only one correlation was detected, i.e., correlation between glucose tolerance and insulin binding to erythrocytes, then under the terguride treatment there appear correlations when basal glycemia is computed versus insulin binding to erythrocytes or to glucose tolerance and/or to triglycerides. Moreover, there is under the terguride treatment correlation insulin binding to erythrocytes versus plasma triglycerides or versus insulinemia. While in SHR/N-cp lean of both sexes and in SHR/N-cp obese males profound changes in the number of statistically significant correlation coefficients were found when controls are compared with animals under the terguride treatment, the different picture we found in SHR/N-cp obese females, i.e., under the terguride treatment correlation basal glycemia versus insulinemia or versus insulin binding to erythrocytes as well as correlation insulin binding to erythrocytes versus insulinemia is present in controls as well as in terguride treated animals. In comparison with controls under terguride only two correlations are missing, i.e., glucose tolerance versus insulinemia or versus insulin binding to erythrocytes.

Involvement of the nervous system in the control of glucose uptake in contracting hindlimb muscle in the rat.

To probe into the possibility of neural control of glucose uptake in contracting peripheral skeletal muscle, rats were divided into two groups: one group is the "normal" (N) group in which the nervous system is intact, and the other is the "denervation" (D) group in which all nerves innervating the hindlimb were cut. The immediate changes in blood glucose (BG) and plasma insulin (PI) levels were measured at the commencement and termination of hindlimb muscle contraction induced with electrical stimulation of the sciatic nerve (distal part in D group). BG in N group temporarily but significantly decreased (p < 0.01) immediately after starting the stimulation, while BG in D group remained unchanged. And the difference between the changes in BG observed in 2 groups was significant (p < 0.03). PI did not change in either group during the sample period. On the other hand, PI temporarily but significantly decreased (p < 0.03) immediately after terminating the stimulation in both groups. The results suggest that the nervous system has some role in controlling glucose uptake in contracting peripheral muscle, at least in the early stage of contraction, that some feedback loop, including the central nervous system, is considered essential for the control, and that some local mechanism promotes an increase in insulin binding to receptors on muscle cells immediately after terminating the contraction.

Effect of age and caloric restriction on insulin receptor binding and glucose transporter levels in aging rats.

We report on the effect of age and chronic caloric restriction (CR) on insulin binding and glucose transporter content in both diaphragm and heart muscle membrane of young (11 months), mid-age (17 months), and old (29 month) ad libitum fed and CR Brown-Norway rats. The control animals received rat chow ad lib and CR animals were allowed 60% of ad libitum food. The CR regimen was initiated at four months of age and the animals were maintained on their respective diets until necropsy. There was no effect of age on insulin binding for either ad libitum or CR animals at each age evaluated. Caloric restriction significantly lowered insulin levels at each age studied when compared to the ad libitum-fed rats. However, CR animals were noted to have increased insulin binding (p < 0.001) compared to ad libitum-fed animals at each age for diaphragm muscle. For the heart, there appeared to be a decreased binding, particularly at higher insulin concentrations, in CR-fed animals. There was no net change in Glut-1 or Glut-4 levels for heart muscle membrane, or Glut-4 levels for diaphragm muscle membrane between ad libitum or CR animals. This data indicates that caloric restriction may have tissue-specific effects for insulin receptor binding, and that the improved insulin sensitivity in CR states is not a result of altered glucose transporter protein content.

Insulin promotes and cyclic adenosine 3',5'-monophosphate impairs functional insertion of insulin receptors in the plasma membrane of rat adipocytes: evidence for opposing effects of tyrosine and serine/threonine phosphorylation.

The aim of the present study was to elucidate events in the plasma membrane (PM) associated with the previously described effect of insulin to rapidly enhance the number of cell surface insulin binding sites in rat adipocytes. [125I]insulin was cross-linked to cell surface insulin receptors of intact cells that had been preincubated with or without insulin. Subsequently prepared PM displayed a approximately 3-fold increase in bound [125I]insulin when cells had been pretreated with 6 nM insulin for 20 min compared to membranes from control cells, and SDS-PAGE with autoradiography showed that this occurred at the insulin receptor alpha-subunit. The magnitude of the effect was similar to that found for insulin binding to intact cells that had been preincubated with insulin. In contrast, the insulin binding capacity in the PM was not affected by prior treatment of cells with insulin when assessed with the addition of [125I]insulin directly to solubilized PM; this suggests an unchanged total number of PM receptors. Thus, the enhancement of cell surface insulin binding capacity produced by insulin is not due to the translocation of receptors, but instead appears to be confined to receptors already present in the PM. The addition of phospholipase C (from Clostridium perfringens), which cleaves PM phospholipids, mimicked the effect of insulin to enhance cell surface binding in adipocytes, and this suggests a pool of cryptic PM receptors. Both the nonmetabolizable cAMP analog N6-monobutyryl cAMP (N6-mbcAMP) and the serine/threonine phosphatase inhibitor okadaic acid abolished the effect of concomitant insulin treatment to increase binding capacity. In contrast, the tyrosine phosphatase inhibitor vanadate increased insulin binding even in the presence of okadaic acid or N6-mbcAMP. The effect of N6-mbcAMP to impair cell surface insulin binding was also evident in the presence of a peptide derived from the major histocompatibility complex type I that effectively impairs receptor internalization, but the amount of PM receptors assessed by immunoblot was unaltered. Taken together, the data suggest that insulin exposure leads to the uncovering of cryptic receptors associated with the PM. It is also suggested that tyrosine phosphorylation promotes this process, whereas enhanced serine phosphorylation, e.g. produced by cAMP, impairs the functional insertion of the receptors, rendering them unable to bind insulin.

Insulin Promotes and Cyclic Adenosine 3',5'-Monophosphate Impairs Functional Insertion of Insulin Receptors in the Plasma Membrane of Rat Adipocytes: Evidence for Opposing Effects of Tyrosine and Serine/Threonine Phosphorylation

The aim of the present study was to elucidate events in the plasma membrane (PM) associated with the previously described effect of insulin to rapidly enhance the number of cell surface insulin binding sites in rat adipocytes. [125I]insulin was cross-linked to cell surface insulin receptors of intact cells that had been preincubated with or without insulin. Subsequently prepared PM displayed a ∼3-fold increase in bound [125I]insulin when cells had been pretreated with 6 nm insulin for 20 min compared to membranes from control cells, and SDS-PAGE with autoradiography showed that this occurred at the insulin receptorα -subunit. The magnitude of the effect was similar to that found for insulin binding to intact cells that had been preincubated with insulin. In contrast, the insulin binding capacity in the PM was not affected by prior treatment of cells with insulin when assessed with the addition of [125I]insulin directly to solubilized PM; this suggests an unchanged total number of PM receptors. Thus, the enhancement of cell surface insulin binding capacity produced by insulin is not due to the translocation of receptors, but instead appears to be confined to receptors already present in the PM. The addition of phospholipase C (from Clostridium perfringens), which cleaves PM phospholipids, mimicked the effect of insulin to enhance cell surface binding in adipocytes, and this suggests a pool of cryptic PM receptors. Both the nonmetabolizable cAMP analog N6-monobutyryl cAMP (N6-mbcAMP) and the serine/threonine phosphatase inhibitor okadaic acid abolished the effect of concomitant insulin treatment to increase binding capacity. In contrast, the tyrosine phosphatase inhibitor vanadate increased insulin binding even in the presence of okadaic acid or N6-mbcAMP. The effect of N6-mbcAMP to impair cell surface insulin binding was also evident in the presence of a peptide derived from the major histocompatibility complex type I that effectively impairs receptor internalization, but the amount of PM receptors assessed by immunoblot was unaltered. Taken together, the data suggest that insulin exposure leads to the uncovering of cryptic receptors associated with the PM. It is also suggested that tyrosine phosphorylation promotes this process, whereas enhanced serine phosphorylation, e.g. produced by cAMP, impairs the functional insertion of the receptors, rendering them unable to bind insulin.

Intracellular insulin binding in Tetrahymena pyriformis

Insulin, a classic vertebrate hormone, produces alterations in cellular metabolism and growth in the ciliate Tetrahymena pyriformis, as well as an increase in insulin binding upon subsequent exposure, a phenomenon known as hormonal imprinting. An antibody to a peptide corresponding to the α-subunit of the human insulin receptor (amino acid residues 657–670) was used to investigate the location and to partially characterize immunoreactive proteins in insulin-exposed and non-insulin-exposed cells (control). Confocal microscopy revealed immunofluorescent labeling of cilia, nuclei, vesicles and an oblong structure of unknown nature. Labeling of nuclei, mitochondria and ciliary microtubules was seen with immunoelectron microscopy. Labeling was absent on the cell and ciliary membranes by immunoelectron microscopy. Polyacrylamide gel electrophoresis revealed several differences in protein composition between control and insulin-exposed ciliary membrane extracts, especially in the 30–50 kDa range. Immunoblotting revealed 2 reactive proteins in whole cell lysates but none were detected in ciliary membrane extracts or wheat germ agglutinin affinity column eluates of T. pyriformis whole cell preparations. Based on these findings it is unlikely that a cell surface structure similar to a mammalian insulin receptor exists in T. pyriformis.

IFN-gamma treatment increases insulin binding and MHC class I expression in erythroleukemia cells.

We have investigated if interferon-gamma (IFN-gamma) treatment of human K562 tumor cells, which upregulates the expression of MHC class I antigens (MHC-I), simultaneously would influence insulin binding. Treatment of K562 cells with recombinant human IFN-gamma for 48 h caused a significant increase of insulin binding at 37 degrees C. Recombinant human tumor necrosis factor-alpha (TNF-alpha) alone had no effect but acted synergistically with IFN-gamma, leading to a two-fold increase of insulin binding. No change in affinity, number of binding sites or cell surface expression of insulin receptors (IR) after IFN-gamma treatment could be detected. The increased insulin binding observed at 37 degrees C was not seen at 4 degrees C, suggesting alteration of insulin internalization. The dose-response curve, as well as the time curve, for the increase in insulin binding after IFN-gamma treatment correlated with enhanced cell surface expression of MHC-I antigens. However, the correlation was not absolute. Our results show that IFN-gamma treatment alone or together with TNF-alpha, can alter the insulin binding to K562 cells without changing the expression or affinity of the IR. This correlates with the effect of IFN-gamma on MHC-I expression. These results support the findings that MHC-I molecules associate and interact with the IR at the cell surface.

Effect of long-term insulin exposure on insulin binding in Tetrahymena pyriformis

Previous studies have indicated that Tetrahymena pyriformis can bind the vertebrate hormone insulin. Conventional microscopic studies were conducted to determine the effect of acute and long-term (LTE) insulin exposure on insulin binding. Stock cultures included cells never exposed to insulin and cultures grown in medium containing 6 mg/ml insulin. Logarithmic cultures were exposed to porcine insulin concentrations of 0 and 6 mg/ml for 1 h (insulin treated, (IT)) after 0, 48 h, 1, 3, or 6 months of LTE insulin exposure. 24 h after the 1 h insulin treatment, the cells were fixed, exposed to porcine insulin (antigen), processed immunocytochemically using a primary antibody to porcine insulin and a secondary antibody immunocytochemistry kit, and examined for staining intensity by video image analysis. Morphological observations confirm that T. pyriformis does bind insulin whether or not the cells have had prior exposure to insulin. IT increases insulin binding (up-regulation) in previously unexposed cells (control, P < 0.01) and produces a further amplification in cells having prior acute exposure (48 h) to insulin (P < 0.01). However, LTE exposure to insulin (1, 3 and 6 months) caused a decrease in insulin binding (down-regulation) after IT (P < 0.01) such that LTE-IT cells were not different from control cells following 1, 3 or 6 months of chronic insulin exposure to insulin. Staining intensity was not different between IT cells and cells cultured with insulin throughout the six month study. Results suggest that insulin binding sites of T. pyriformis are subject to regulatory processes similar to those of metazoans.

Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cell-surface GLUT4 as measured by photoaffinity labeling.

(+/-)-5-([4-[2-Methyl-2(pyridylamino)ethoxy]phenyl]methyl) 2,4-thiazolidinedione (BRL 49653) is a new potent antidiabetic agent that improves insulin sensitivity in animal models of NIDDM. In C57BL/6 obese (ob/ob) mice, BRL 49653, included in the diet for 8 days, improved glucose tolerance. The half-maximal effective dose was 3 mumol/kg diet, which is equivalent to approximately 0.1 mg/kg body wt. Improvements in glucose tolerance were accompanied by significant reductions in circulating triacylglycerol, nonesterified fatty acids, and insulin. The insulin receptor number of epididymal white adipocytes prepared from obese mice treated with BRL 49653 (30 mumol/kg diet) for 14 days was increased twofold. The affinity of the receptor for insulin was unchanged. In the absence of added insulin, the rates of glucose transport in adipocytes from untreated and BRL 49653-treated obese mice were similar. Insulin (73 nmol/l) produced only a 1.5-fold increase in glucose transport in adipocytes from control obese mice, whereas after BRL 49653 treatment, insulin stimulated glucose transport 2.8-fold. BRL 49653 did not alter the sensitivity of glucose transport to insulin. The increase in insulin responsiveness was accompanied by a 2.5-fold increase in the total tissue content of the glucose transporter GLUT4. Glucose transport in adipocytes from lean littermates was not altered by BRL 49653. To establish the contribution of changes in glucose transporter trafficking to the BRL 49653-mediated increase in insulin action, the cell-impermeant bis-mannose photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos++ +-4-yloxy) -2-[2-3H]-propylamine was used to measure adipocyte cell-surface-associated glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)