Modulation of expression of insulin and IGF-I receptor by Epstein-Barr virus and its gene products LMP and EBNA-2 in lymphocyte cell lines

The receptors for insulin and insulin-like growth factor I (IGF-I) are two closely related integral membrane glycoproteins involved in signalling of cell growth and metabolism. We have used the unique paradigm of pairs of Burkitt lymphoma cell lines (BLO2, BL30, BL41) with or without Epstein-Barr Virus (EBV) infection and cells transfected with EBV-related genes to examine effects of EBV on expression of these receptors at the gene and protein functional level. In BL30 and BL41 cells, EBV infection increased surface insulin binding and total receptor number by 2-and 18-fold, respectively. By contrast, EBV infection decreased total IGF-I receptors by 29 to 87% in all three cell lines. In general, there was a correlation between total receptor concentration and the level of insulin or IGF-I receptor mRNAs, although in one cell line insulin binding increased while receptor mRNA levels decreased slightly, suggesting posttranslational effects. BL41 cells transfected with a vector expressing the EBV latent membrane protein (LMP) exhibited a 2.6- to 3.2-fold increase in insulin receptor expression, whereas cells transfected with EBNA-2 (one of the EBV nuclear antigens) alone exhibited no effect. However, EBNA-2 appears to be required for the EBV effect on insulin receptor expression since cells infected with a mutant virus, P3JHRI, which lacks the EBNA-2 gene failed to show an increase in insulin receptor number. These data indicate that EBV infection of lymphocytes increases expression of insulin receptors while simultaneously decreasing expression of IGF-I receptors. The magnitude and sometimes even the direction of change, depends on host cell factors. A maximal increase in insulin receptors appears to require the coordinate action of several of the EBV proteins including LMP and EBNA-2. © 1993 Wiley-Liss, Inc.     

Functional alterations in pancreatic beta cells as a factor in virus-induced hyperglycemia in mice

Alterations in the functional capacity of pancreatic beta cells appear to contribute to coxsackievirus B4-induced, long-term hyperglycemia in mice. Mice infected with prototype B4 or its diabetogenic E2 variant were monitored for abnormalities in sugar metabolism (by the glucose tolerance test), for total protein and insulin synthesis in intact beta cells, for alterations in beta cell proteins, and for virus replication. The infected mice were hypoglycemic at 72 h postinfection and hyperglycemic at 6 weeks. At 8 weeks postinfection, few of the prototype- but most of the E2-infected mice remained hyperglycemic. Total protein and synthesis of immunoprecipitable insulin decreased during early infection. At 8 weeks postinfection, insulin synthesis in the prototype-infected mice increased almost to the level of control mice. Although insulin synthesis increased likewise in the E2-infected mice, it remained well below the control level. Two-dimensional gel electrophoresis revealed the disappearance of many cellular proteins in beta cells from E2-infected mice but of very few in cells from prototype-infected mice at 72 h postinfection. Many of the disappearing proteins reappeared gradually in the E2-infected group. Infectious virus was recovered from the infected beta cells only at 72 h postinfection. Functional impairment in these cells appears to be a factor in virus-induced hyperglycemia.     

Obese mice have higher insulin receptor levels in the hepatocyte cell nucleus following insulin stimulation in vivo with an oral glucose meal.

Internalization of the insulin receptor occurs following insulin binding at the cell surface, which serves to attenuate the insulin signal as well as modulate the number of surface insulin receptors. Obese animals exhibit decreased cell surface insulin receptor number as well as defects in insulin receptor internalization and processing. The insulin receptor may also translocates to the nucleus of hepatocytes and adipocytes following stimulation of cells with insulin. The objective of this study was to determine if insulin receptor trafficking to the hepatocyte cell nucleus could be observed in vivo and whether this process was altered in obese compared to lean mice. Mice were fasted for 12 h to reduce serum insulin to basal levels. Animals were then given an oral meal of glucose to stimulate the binding of insulin to receptor in vivo. Hepatocyte plasma membrane and nuclei were fractionated to purity following the glucose meal. Levels of insulin receptor were determined using insulin binding assays and a Western blotting assay using anti-insulin receptor antibody. As the amount of serum insulin increased following the glucose meal, a corresponding increase in nuclear insulin binding occurred in lean animals but not obese animals (P<0.05). Following the glucose meal, insulin receptor detected in the cell nucleus was increased in obese compared to lean mice (P<0.05). Thus insulin receptor translocation to the nucleus was demonstrated in vivo following a glucose meal in hepatocytes of both lean and obese animals. It is suggested that serum hyperglycemia and hyperinsulinemia in obese mice increased translocation of the insulin receptor to the nucleus.     

Alteration of insulin receptor kinase in obese, insulin-resistant mice

We have studied the properties of muscle insulin receptors obtained from genetically or experimentally-induced obese mice that are both insulin-resistant. Insulin receptors, partially purified by wheat germ agglutinin--agarose chromatography, were studied in a cell-free system for autophosphorylation, for their ability to phosphorylate a synthetic glutamate--tyrosine copolymer and for their binding characteristics. Insulin receptor number was decreased by 25% in muscles from obese mice without any change in their binding affinity. The insulin stimulatory action on its beta-subunit receptor phosphorylation was diminished in preparations from genetically- or experimentally-induced obese mice to a higher degree than the decrease in insulin receptor number. HPLC analysis of the phosphopeptides generated by trypsin treatment of the labeled receptor beta-subunit was identical in lean and obese mice. Similar alteration of the kinase activity was found in obese mice when the phosphorylation of casein or polyglutamate--tyrosine was measured. Trypsin treatment of the receptor preparations was less effective in stimulating the kinase activity in obese mice than in lean mice. These results suggest that the defect in insulin receptor kinase activity reflects an alteration in the transmission of the message from the alpha- to the beta-subunit or an impairment of the enzyme functioning by environmental conditions.     

Caveolin-3 knockout mice show increased adiposity and whole body insulin resistance, with ligand-induced insulin receptor instability in skeletal muscle

Caveolin-3 (Cav-3) is expressed predominantly in skeletal muscle fibers, where it drives caveolae formation at the muscle cell's plasma membrane. In vitro studies have suggested that Cav-3 may play a positive role in insulin signaling and energy metabolism. We directly address the in vivo metabolic consequences of genetic ablation of Cav-3 in mice as it relates to insulin action, glucose metabolism, and lipid homeostasis. At age 2 mo, Cav-3 null mice are significantly larger than wild-type mice, and display significant postprandial hyperinsulinemia, whole body insulin resistance, and whole body glucose intolerance. Studies using hyperinsulinemic-euglycemic clamps revealed that Cav-3 null mice exhibited 20% and 40% decreases in insulin-stimulated whole body glucose uptake and whole body glycogen synthesis, respectively. Whole body insulin resistance was mostly attributed to 20% and 40% decreases in insulin-stimulated glucose uptake and glucose metabolic flux in the skeletal muscle of Cav-3 null mice. In addition, insulin-mediated suppression of hepatic glucose production was significantly reduced in Cav-3 null mice, indicating hepatic insulin resistance. Insulin-stimulated glucose uptake in white adipose tissue, which does not express Cav-3, was decreased by 70% in Cav-3 null mice, suggestive of an insulin-resistant state for this tissue. During fasting, Cav-3 null mice possess normal insulin receptor protein levels in their skeletal muscle. However, after 15 min of acute insulin stimulation, Cav-3 null mice show dramatically reduced levels of the insulin receptor protein, compared with wild-type mice treated identically. These results suggest that Cav-3 normally functions to increase the stability of the insulin receptor at the plasma membrane, preventing its rapid degradation, i.e., by blocking or slowing ligand-induced receptor downregulation. Thus our results demonstrate the importance of Cav-3 in regulating whole body glucose homeostasis in vivo and its possible role in the development of insulin resistance. These findings may have clinical implications for the early diagnosis and treatment of caveolinopathies. limb girdle muscular dystrophy; glucose intolerance; hyperinsulinemia; insulin receptor degradation     

Whole body hyperthermia of rats decreases insulin binding to erythrocytes

125I-insulin binding to rat erythrocytes was studied to investigate the effect of whole body hyperthermia on the insulin receptor. Heat treatment of rats at 42 degrees C for 15 min caused a significant decrease (48.7% of control) in 125I-insulin binding to rat erythrocytes. Scatchard analysis showed that the decreased binding resulted from a decrease in the number of the insulin receptors rather than from a decrease in receptor affinity. The decreased receptor number for insulin showed no evidence of recovery, 2 h and 8 h after the hyperthermia. Plasma insulin levels remained lower than the control, up to 8 h after the hyperthermia, whereas plasma glucose, which decreased immediately after the hyperthermia, increased higher than the control, 8 h after the hyperthermia. The low plasma insulin level and decreased number of insulin receptor are believed to be possible factors for the elevation of plasma glucose.     

Epidermal growth factor binding, stimulation of phosphorylation, and inhibition of gluconeogenesis in rat proximal tubule

Epidermal growth factor and insulin share many biological activities, including stimulation of cell proliferation, ion flux, glycolysis, fatty acid and glycogen synthesis, and activation of receptor-linked tyrosine kinase activity. In the kidney, insulin has been shown to regulate transport processes and inhibit gluconeogenesis in the proximal tubule. Since the kidney represents a major source of EGF, the present studies investigated whether proximal tubule contained EGF receptors, whether EGF receptors were localized to apical or basolateral membranes, and whether EGF receptor activation participated in the regulation of an important proximal tubule function, gluconeogenesis. Specific EGF receptors were demonstrated in the basolateral membrane of proximal tubule. Following incubation with 125I EGF, basolateral membranes demonstrated equilibrium binding at 4 degrees C and 23 degrees C. There was 78 +/- 2% specific binding (n = 13). The dissociation constant (Kd) was 1.5 x 10(-9) M and maximal binding was 44 fmol/mg protein. There was ninefold more specific binding to proximal tubule basolateral membrane than to brush border membrane. In basolateral, but not brush border membranes, EGF induced phosphorylation of the tyrosine residues of intrinsic membrane proteins, including a 170 kDa protein, corresponding to the EGF receptor. In the presence of the gluconeogenic substrates, alanine, lactate, and succinate, proximal tubule suspensions synthesized glucose. EGF inhibited glucose production in a concentration-dependent manner over a concentration range of 3 x 10(-11) to 3 x 10(-9) M. In addition, EGF inhibited angiotensin II-stimulated glucose production in the proximal tubule suspensions. EGF did not significantly increase net glucose metabolism nor decrease cellular ATP concentrations. Therefore, these studies demonstrated that rat proximal tubule contained specific receptors for EGF that were localized to the basolateral membrane and linked to tyrosine kinase activity. EGF significantly inhibited proximal tubule glucose production without significantly increasing net glucose consumption.     

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.     

Involvement of glycoconjugates in insulin-receptor interactions Studies in liver plasma membranes of control and diabetic mice

The involvement of glycoconjugates in the insulin-receptor interactions in mouse liver is tested by digestions of membranes with various enzymes. Trypsin decreased the binding of [¹²⁵I]insulin to liver membranes. After digestion with β-galactosidase no “high affinity” receptor sites could be detected. The effects observed with plant lectins confirm the involvement of galactoconjugates in the insulin binding process. Sophora japonica and Ricinus communis lectins (with galactose specificity) and concanavalin A largely inhibit the binding process of insulin and those effects concern the “high affinity” receptor sites. Other lectins (wheat germ agglutinin, Dolichos) and enzymes (α-l-fucosidase, $\text{β-N-}\text{acetyl-hexosaminidase and neuraminidase}$) are without effect on insulin binding.Comparative studies performed on diabetic mouse liver membrane (KK mice), previously characterized by decreased number of insulin receptors, are in good agreement with qualitatively similar receptor sites in both non-diabetic (control) and diabetic mice. Effects of enzymes and lectins yielded same results as compared to control membranes. Plasma membrane proteins and glycoproteins in both types of mouse are indistinguishable with respect to enzymic and chemical analysis. Sodium dodecyl sulphate acrylamide gel electrophoresis shows identical patterns. Moreover, the decrease in the number of insulin receptors is easily reversed with diet restriction. These data are consistent with the similarity of receptor sites in control and diabetic liver membrane.     

Down regulation of masked and unmasked insulin receptors in the liver of transgenic mice expressing bovine growth hormone gene.

The interaction of insulin with its receptor was studied in microsomes from livers of transgenic mice expressing the bovine growth hormone gene with mouse metallothionein-1 promoter (MT/bGH) and in their normal (non-transgenic) littermates. Specific binding of 125I-insulin was detected in hepatic microsomes from normal and transgenic mice with an apparent Kd of 8 and 200 nM, for high and low affinity sites, respectively. The transgenic MT/bGH mice had a marked hyperinsulinism without significant elevation of plasma glucose levels. Under identical conditions of preparation and incubation, microsomes from the transgenic male and female mice bound 39% and 34% less insulin than those from their litter mates. Scatchard's analysis indicates that this decrease in binding is due to a decrease in the number of receptor sites. In contrast to the marked decrease in insulin binding to unmasked receptors, the levels of masked (also called cryptic) insulin receptors were similar (or slightly increased) in transgenic mice microsomes as compared to those of their normal litter mates.     

The insulin receptor--single function and dual effect.

Net effects of insulin on glucose entry, metabolism and other cellular processes have been well documented over the past 30-40 years. Although it is known that insulin binds to a specific cell membrane receptor protein which undergoes autophosphorylation and tyrosine kinase activation, the individual reactions following receptor activation that cause the metabolic changes remain unknown. It is well documented that the isolated insulin receptor has a high degree of basal autophosphorylation capacity and externally directed tyrosine kinase. There is also evidence that some in vivo autophosphorylation can take place in the total absence of insulin. If receptor activity does exist in the absence of insulin, then receptor function needs to be reanalyzed. It will be proposed here that the insulin binding membrane protein functions mainly to inhibit glucose transport under low physiological levels of insulin. Evidence of basal receptor enzymatic activity in the absence of insulin supports this theory. Under metabolically sufficient conditions, enough insulin receptors are functionally active to interact with the glucose transport system in an inhibitory manner, providing membrane control of internal glucose metabolism. Insulin acts by aggregating this inhibitory system. If inhibitory insulin receptors are aggregated following insulin elevation, their inhibitory action is prevented and glucose transport increases. This increase in transport will be in direct proportion to the temporal inhibitory level of the receptor and to the area of the cell membrane cleared of their inhibitory effect. When insulin receptor protein is confined to small areas of the cell membrane through aggregation, any potential inhibitory function is negated and glucose entry increases dramatically. This is the classical insulin effect. Both of these concepts were suggested 37 years earlier. Randle & Smith (1957, Biochem. Biophys. Acta 25, 442; 1958, Biochem. J. 70, 490) proposed that the internal supply of energy rich compounds limited glucose entry and that the effect of insulin was to inhibit this process which was inhibiting glucose entry. The present report provides a mechanism for this.     

The insulin-like effect of sodium vanadate on adipocyte glucose transport is mediated at a post-insulin-receptor level.

Sodium vanadate has several insulin-like effects. To determine whether vanadate acts via the insulin receptor, I investigated the effect of vanadate on glucose transport (2-deoxyglucose uptake) in adipocytes that had been treated to decrease the number of insulin receptors. Trypsin (100 micrograms/ml) caused greater than 95% loss of 125I-insulin binding and rendered glucose transport resistant to both insulin and an anti-insulin-receptor antibody. However, vanadate caused an 8-fold increase in the transport rate [EC50 (concn. giving 50% of maximum effect) 0.2 mM] in both control and trypsin-treated cells, demonstrating that the insulin receptor does not have to be intact for vanadate to stimulate glucose transport. Insulin receptors were depleted by treatment of adipocytes with insulin (100 ng/ml) in the presence of Tris (which blocks receptor recycling). A 2 h treatment caused 60% loss of receptors, and a shift to the right in the dose-response curve for insulin stimulation of glucose transport (EC50 0.3 ng of insulin/ml in controls, 1.2 ng/ml in treated cells). The response to vanadate was again unaffected. Treatment with insulin for 4 h caused a 67% decrease in insulin binding and, in addition to the rightward shift in the insulin dose-response curve, a decrease in basal and maximal transport rates (which cannot be explained by decreased insulin receptor number). The EC50 of vanadate was again equal in control and treated cells, but glucose transport in the presence of a maximally effective concentration of vanadate (1 mM) was decreased. I conclude that the effect of vanadate on glucose transport is independent of the insulin receptor. Induction of a post-receptor defect (which may be a decrease in the total number of cellular glucose transporters) by prolonged exposure to insulin decreases the potency of a maximally effective concentration of vanadate. The findings demonstrate that vanadate stimulates glucose transport by an effect at a level distal to the insulin receptor.     

In L6 skeletal muscle cells, glucose induces cytosolic translocation of protein kinase C-alpha and trans-activates the insulin receptor kinase.

In L6 skeletal muscle cells expressing human insulin receptors (L6(hIR)), exposure to 25 mM glucose for 3 min induced a rapid 3-fold increase in GLUT1 and GLUT4 membrane translocation and glucose uptake. The high glucose concentration also activated the insulin receptor kinase toward the endogenous insulin receptor substrates (IRS)-1 and IRS-2. At variance, in L6 cells expressing kinase-deficient insulin receptors, the exposure to 25 mM glucose elicited no effect on glucose disposal. In the L6(hIR) cells, the acute effect of glucose on insulin receptor kinase was paralleled by a 2-fold decrease in both the membrane and the insulin receptor co-precipitated protein kinase C (PKC) activities and a 3-fold decrease in receptor Ser/Thr phosphorylation. Western blotting of the receptor precipitates with isoform-specific PKC antibodies revealed that the glucose-induced decrease in membrane- and receptor-associated PKC activities was accounted for by dissociation of PKCalpha but not of PKCbeta or -delta. This decrease in PKCalpha was paralleled by a similarly sized increase in cytosolic PKCalpha. In intact L6(hIR) cells, inhibition of PKCalpha expression by using a specific antisense oligonucleotide caused a 3-fold increase in IRS phosphorylation by the insulin receptor. This effect was independent of insulin and accompanied by a 2.5-fold increase in glucose disposal by the cells. Thus, in the L6 skeletal muscle cells, glucose acutely regulates its own utilization through the insulin signaling system, independent of insulin. Glucose autoregulation appears to involve PKCalpha dissociation from the insulin receptor and its cytosolic translocation.     

Ⅱ型糖尿病大鼠海马Alzheimer病样Tau蛋白过度磷酸化修饰及机制探讨

Tau蛋白过度磷酸化是Alzheimer病 (Alzheimer′s disease, AD) 的一个重要特征.本研究检测了Ⅱ型糖尿病大鼠海马tau蛋白磷酸化水平,对其形成机制进行探讨. 以同龄正常Wistar大鼠作为对照,高脂高蛋白高糖饮食加小剂量链脲佐菌素（streptozotocin,STZ）注射诱导造Ⅱ型糖尿病模型（T2DM组）.放免法检测血浆胰岛素;葡萄糖氧化酶法检测血浆葡萄糖;蛋白质印迹技术检测各组大鼠海马内总tau蛋白、tau蛋白上部分位点磷酸化、神经细胞膜上胰岛素受体及葡萄糖转运子3（glucose transport 3,GLUT3）水平;表面等离子共振技术（surface plasmon resonance, SPR）检测细胞膜上胰岛素受体与血浆胰岛素结合力;γ32-P标记的ATP和特异性底物肽检测海马内胰岛素信号传导系统中的关键酶糖原合酶激酶-3β（glycogen synthase kinase-3β, GSK-3β）活性.结果显示,T2DM组血浆血糖、血浆胰岛素及运用HOMA-IR公式计算的胰岛素抵抗指数显著高于对照组.蛋白质印迹结果显示两组大鼠海马回总tau蛋白水平无差异;T2DM组中tau蛋白在Ser199、Thr212、Ser214、Thr217、Ser396及Ser422位点上的磷酸化水平均显著高于对照组;T2DM组海马神经细胞膜上胰岛素受体水平及与胰岛素结合的功能均显著低于对照组;GSK-3β活性检测结果显示,T2DM组大鼠模型海马回中GSK-3β活性明显增高.研究结果表明,Ⅱ型糖尿病中由于胰岛素抵抗导致GSK-3β激活从而出现AD样tau蛋白的过度磷酸化,葡萄糖代谢紊乱也可能在tau蛋白的过度磷酸化起一定作用.     

Nitric oxide: the "second messenger" of insulin

Incubation of various tissues, including heart, liver, kidney, muscle, and intestine from mice and erythrocytes or their membrane fractions from humans, with physiologic concentration of insulin resulted in the activation of a membrane-bound nitric oxide synthase (NOS). Activation of NOS and synthesis of NO were stimulated by the binding of insulin to specific receptors on the cell surface. A Lineweaver-Burk plot of the enzymatic activity demonstrated that the stimulation of NOS by insulin was related to the decrease in the Km for L-arginine, the substrate for NOS, with a simultaneous increase of Vmax. Addition of NG-nitro-L-arginine methyl ester (LNAME), a competitive inhibitor of NOS, to the reaction mixture completely inhibited the hormone-stimulated NO synthesis in all tissues. Furthermore, NO had an insulin-like effect in stimulating glucose transport and glucose oxidation in muscle, a major site for insulin action. Addition of NAME to the reaction mixture completely blocked the stimulatory effect of insulin by inhibiting both NO production and glucose metabolism, without affecting the hormone-stimulated tyrosine or phosphatidyl-inositol 3-kinases of the membrane preparation. Injection of NO in alloxan-induced diabetic mice mimicked the effect of insulin in the control of hyperglycemia (i.e., lowered the glucose content in plasma). However, injection of NAME before the administration of insulin to diabetic-induced and nondiabetic mice inhibited not only the insulin-stimulated increase of NO in plasma but also the glucose-lowering effect of insulin.     

Hypoglycaemia induced by Trichinella infection is due to the increase of glucose uptake in infected muscle cells

The present study investigates how Trichinella infection induces host hypoglycaemia and explores a potential relationship between infection and the insulin signalling pathway. The results showed that mice infected with Trichinella spiralis or Trichinella pseudospiralis exhibited a temporary decrease in blood glucose level between 8 and 28 days p.i. and the kinetics of the glucose levels corresponded to the process of muscle larval growth and development. Histochemical results showed that glycogen accumulation increased in infected muscle cells during the period of hypoglycaemia. Analysis of gene expression profiles with quantitative PCR demonstrated that insulin signalling pathway-related genes, such as insulin receptor (IR), insulin receptor substance 1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and V-akt murine thymoma viral oncogene homologue 2 (Akt2) were up-regulated in infected muscle cells during infection and these expression changes correlated with the kinetics of blood glucose level, glycogen accumulation and the process of larval growth and development in infected muscle cells. Western blot analysis clarified that the expression of IR and Akt2 proteins increased in muscle tissues infected with both species of Trichinella. This study suggests that hypoglycaemia induced by Trichinella infection is the result of an increase in glucose uptake by infected muscle cells via up-regulation of insulin signalling pathway factors.     

Mechanism of methaemoglobin reduction by human erythrocytes.

The effect of alterations to the insulin receptor on the insulin sensitivity of isolated adipocytes was studied. Receptor changes were induced by treatment of adipocytes with either phospholipase C or trypsin. After enzyme treatment, binding of insulin to insulin receptors and insulin-mediated glucose metabolism were examined. Exposure of adipocytes to phospholipase C (2 units/ml) significantly increased insulin binding to the cells, but destroyed the ability of the cells to oxidize glucose. After treatment with trypsin (500 micrograms/ml) for 5 min, insulin binding to the adipocytes was significantly increased. This was shown to be due to an increase in insulin-receptor affinity. Metabolic studies showed that trypsin treatment led to an increase in basal glucose transport but markedly decreased the response to insulin at all concentrations tested. Adipocytes treated with trypsin showed no significant difference in basal glucose oxidation rates when compared with controls, but were less sensitive to insulin at low insulin concentrations, and showed a decreased maximum response at high insulin concentrations. In conclusion, these findings indicate a dissociation between induced changes in binding of insulin to insulin receptors and subsequent hormone action. The importance of post-receptor events in the biological action of insulin is highlighted.     

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.     

Functional Properties of the Subtype of Insulin Receptor Found on Neurons

In this report, we have examined the structure, regulation, and function of insulin receptors in cultured neurons from fetal chicken brain. The apparent molecular weight of the alpha-subunit of neuronal insulin receptors, analyzed by photoaffinity labeling and sodium dodecyl sulfate gel electrophoresis under reducing conditions, was 115,000. The number of insulin receptors in the cultures increased from day 2 to day 4 during a period of extensive process formation. After 5 days in culture, there were approximately 40,000 high-affinity insulin receptors per neuron. When neurons were photoaffinity labeled at 16 degrees C and then warmed to 37 degrees C for 30 min, approximately 40% of the cell-surface receptors were recovered in the intracellular, trypsin-insensitive pool. Chronic exposure of neurons to insulin (100 ng/ml) resulted in a time-dependent loss of neuronal insulin receptors with a maximal decrease of 50% after 24 h. Insulin had no effect on glucose transport, glucose oxidation, or glycogen synthase activity in neurons. On the other hand, insulin supported the growth and differentiation of a fraction of neurons isolated from chick forebrain. We conclude that (1) cultured neurons from fetal chicken brain express the same subtype of insulin receptor previously identified in adult rat and human brain, (2) the neuronal subtype of insulin receptor undergoes internalization and down-regulation in response to insulin, and (3) neuronal insulin receptors do not acutely regulate glucose metabolism but mediate growth in neurons.