Effect of hyperglycemia on insulin receptor signaling in the cultured retinal Müller glial cells

Hyperglycemia and impaired insulin signaling are considered as major factors in the retinal pathology in diabetic retinopathy (DR). Numerous reports support that these two factors damage retinal glial as well as neuronal cells early in diabetes. However, it is not known whether diabetic induced hyperglycemia causes a depression to the insulin signaling. In this study we utilized a well characterized cultured Muller cells (TR-MUL) where we found a high expression of insulin receptor molecules. TR-MUL Cells were treated with high glucose, glutamate and hydrogen peroxide, and activated with insulin. Following treatments, cell lysates were analyzed by immunoblotting experiments for insulin receptor (IRβ) and insulin receptor substrate (IRS1). In addition, cell lysates were immunoprecipitated using antibodies against insulin receptor proteins to analyze tyrosine phosphorylation and serine phosphorylation of insulin receptor proteins. Results indicate that hyperglycemia did not affect the expression of insulin receptor proteins in cultured TR-MUL cells. Although, hyperglycemia seems to inhibit the interaction between IRS1 and IRβ. Hydrogen peroxide increased the tyrosine phosphorylation of insulin receptor proteins but excess glutamate could not affect the insulin receptor proteins indicating that glutamate may not cause oxidative stress in TR-MUL cells. Hyperglycemia lowered serine phosphorylation of IRS^ser632 and IRS^ser1101 however, IRS^ser307 was not affected. Thus, hyperglycemia may not affect insulin signaling through tyrosine phosphorylation of insulin receptor proteins but may inhibit the interactions between insulin receptor proteins. Hyperglycemia induced phosphorylation of various serine residues of IRS1 and their influence on insulin signaling needs further investigation in TR-MUL cells.     

Cytoplasmic juxtamembrane region of the insulin receptor: a critical role in ATP binding, endogenous substrate phosphorylation, and insulin-stimulated bioeffects in CHO cells.

We have expressed in CHO cells a mutant receptor (IR delta 960) from which 12 amino acids in the juxtamembrane region (A954-D965), including Tyr960, have been deleted. The mutant receptor bound insulin normally but exhibited an increased Km for ATP during autophosphorylation. Upon prolonged incubation in vitro, or at high ATP concentrations such as those observed in vivo, autophosphorylation of IR delta 960 was similar to wild type, and the in vitro phosphotransferase activity of the autophosphorylated IR delta 960 was normal. These results suggest that the deletion did not cause a nonspecific structural disruption of the catalytic domain of IR delta 960. In vivo autophosphorylation of the IR delta 960 receptor was reduced by 30% after 2 min of insulin stimulation and was similar to the wild-type receptor after 30 min of insulin stimulation. However, the mutant receptor was defective in insulin-stimulated tyrosyl phosphorylation of the endogenous substrate pp185. In addition, IR delta 960 was deficient in mediating insulin stimulation of glycogen and DNA synthesis. Thus, autophosphorylation of the insulin receptor is necessary but not sufficient for signal transmission. These data extend the hypothesis that the cytoplasmic juxtamembrane region of the insulin receptor is important for its interactions with ATP, intracellular substrates, and other proteins and is broadly necessary for biological signal transmission.     

Berberine inhibits PTP1B activity and mimics insulin action

Type 2 diabetes patients show defects in insulin signal transduction that include lack of insulin receptor, decrease in insulin stimulated receptor tyrosine kinase activity and receptor-mediated phosphorylation of insulin receptor substrates (IRSs). A small molecule that could target insulin signaling would be of significant advantage in the treatment of diabetes. Berberine (BBR) has recently been shown to lower blood glucose levels and to improve insulin resistance in db/db mice partly through the activation of AMP-activated protein kinase (AMPK) signaling and induction of phosphorylation of insulin receptor (IR). However, the underlying mechanism remains largely unknown. Here we report that BBR mimics insulin action by increasing glucose uptake ability by 3T3-L1 adipocytes and L6 myocytes in an insulin-independent manner, inhibiting phosphatase activity of protein tyrosine phosphatase 1B (PTP1B), and increasing phosphorylation of IR, IRS1 and Akt in 3T3-L1 adipocytes. In diabetic mice, BBR lowers hyperglycemia and improves impaired glucose tolerance, but does not increase insulin release and synthesis. The results suggest that BBR represents a different class of anti-hyperglycemic agents.     

Distribution of actin and tropomyosin in Cryptosporidium muris

Actin and tropomyosin of Cryptosporidium muris were localized by immunogold labeling. Two kinds of antibodies for actin labeling were used. The polyclonal antibody to skeletal muscle (chicken back muscle) actin was labeled on the pellicle and cytoplasmic vacuoles of parasites. The feeder organelle has showed a small amount of polyclonal actin antibody labeling as well. Whereas the monoclonal antibody to smooth muscle (chicken gizzard muscle) actin was chiefly labeled on the filamentous cytoplasm of parasites. The apical portion of host gastric epithelial cell cytoplasm was also labeled by smooth muscle actin together. The polyclonal antibody to tropomyosin was much more labeled at C. muris than host cells, so it could be easily identified even with low magnification (×2,000). The tropomyosin was observed along the pellicle, cytoplasmic vacuoles, and around the nucleus also. The skeletal muscle type actin seems to play a role in various cellular functions with tropomyosin in C. muris; on the other hand, the smooth muscle type actin was located mainly on the filamentous cytoplasm and supported the parasites'' firm attachment to host cells. Tropomyosin on the pellicle was thought to be able to stimulate the host as a major antigen through continuous shedding out by the escape of sporozoites or merozoites from their mother cells.     

Insulin receptor signaling regulates actin cytoskeletal organization in developing photoreceptors

The insulin receptor (IR) and IR signaling proteins are widely distributed throughout the CNS. IR signaling provides a trophic signal for transformed retinal neurons in culture and we recently reported that deletion of IR in rod photoreceptors by Cre/ lox system resulted in stress-induced photoreceptor degeneration. These studies suggest a neuroprotective role of IR in rod photoreceptor cell function. However, there are no studies available on the role of insulin-induced IR signaling in the development of normal photoreceptors. To examine the role of insulin-induced IR signaling, we analyzed cultured neuronal cells isolated from newborn rodent retinas. In insulin-lacking cultures, photoreceptors from wild-type rat retinas exhibited an abnormal morphology with a wide axon cone and disorganization of the actin and tubulin cytoskeleton. Photoreceptors from IR knockout mouse retinas also exhibited a similar abnormal morphology. A novel finding in this study was that addition of docosahexaenoic acid, a photoreceptor trophic factor, restored normal axonal outgrowth in insulin-lacking cultures. These data suggest that IR signaling pathways regulate actin and tubulin cytoskeletal organization in photoreceptors; they also imply that insulin and docosahexaenoic acid activate at least partially overlapping signaling pathways that are essential for the development of normal photoreceptors.     

Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor

Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k(cat) and a 15-fold-lower K(m ATP) although K(m peptide) was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.     

Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation

Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors.     

Characterization of insulin signaling in rat retina in vivo and ex vivo

Insulin receptor (IR) signaling cascades have been studied in many tissues, but retinal insulin action has received little attention. Retinal IR signaling and activity were investigated in vivo in rats that were freely fed, fasted, or injected with insulin by phosphotyrosine immunoblotting and by measuring kinase activity. A retina explant system was utilized to investigate the IR signaling cascade, and immunohistochemistry was used to determine which retinal cell layers respond to insulin. Basal IR activity in the retina was equivalent to that in brain and significantly greater than that of liver, and it remained constant between freely fed and fasted rats. Furthermore, IR signaling increased in the retina after portal vein administration of supraphysiological doses of insulin. Ex vivo retinas responded to 10 nM insulin with IR beta-subunit (IRbeta) and IR substrate-2 (IRS-2) tyrosine phosphorylation and AktSer473 phosphorylation. The retina expresses mRNA for all three Akt isoforms as determined by in situ hybridization, and insulin specifically increases Akt-1 kinase activity. Phospho-AktSer473 immunoreactivity increases in retinal nuclear cell layers with insulin treatment. These results demonstrate that the retinal IR signaling cascade to Akt-1 possesses constitutive activity, and that exogenous insulin further stimulates this prosurvival pathway. These findings may have implications in understanding normal and dysfunctional retinal physiology.     

EphA4 catalytic activity causes inhibition of RhoA GTPase in Xenopus laevis embryos

The Eph family of receptor tyrosine kinases is involved in limiting cell and tissue interactions via a repulsive mechanism. The mechanism of repulsion involves reorganizing the actin cytoskeleton, but little is known of the molecular components that connect the receptor to the actin cytoskeleton. Recent studies in retinal ganglion cells have demonstrated that EphA4 activates the small GTPase Rho. We have investigated the involvement of Rho in signaling downstream from EphA4. As a model system, we have used a chimeric receptor called EPP that we express and activate in early Xenopus embryos. Previous studies demonstrated that EPP activation leads to loss of cell-cell adhesion and change in cell shape, plus loss of aspects of cell polarity in epithelial cells, such as apical microvilli and the apical/basolateral boundary. In this study, we show that injecting inhibitors of Rho GTPases into early Xenopus embryos produces a phenotype very similar to that resulting from EPP activation. More importantly, expression of a constitutively active form of Xenopus RhoA (XRhoA) concurrent with activated EPP rescued embryos from the loss of cell-cell adhesion and change in cell shape associated with EPP. These data argue that, in contrast to the case in retinal ganglion cells, EphA4 in early Xenopus embryos acts to inhibit RhoA, suggesting that this receptor may regulate Rho differently (and therefore affect the cytoskeleton differently) in neuronal and non-neuronal cells. Furthermore, overexpression of ephexin, a novel guanine nucleotide exchange factor for Rho family GTPases, also blocks EPP-induced dissociation. This suggests that EphA4, which has been demonstrated to activate ephexin in cultured neuronal cells, may also target Rho GTPase via an ephexin-independent pathway.     

Sterol structure dependence of insulin receptor and insulin-like growth factor 1 receptor activation

The plasma membrane is a dynamic environment with a complex composition of lipids, proteins, and cholesterol. Areas enriched in cholesterol and sphingolipids are believed to form lipid rafts, domains of highly ordered lipids. The unique physical properties of these domains have been proposed to influence many cellular processes. Here, we demonstrate that the activation of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) depends critically on the structures of membrane sterols. IR and IGF1R autophosphorylation in vivo was inhibited by cholesterol depletion, and autophosphorylation was restored by the replacement with exogenous cholesterol. We next screened a variety of sterols for effects on IR activation. The ability of sterols to support IR autophosphorylation was strongly correlated to the propensity of the sterols to form ordered domains. IR autophosphorylation was fully restored by the incorporation of ergosterol, dihydrocholesterol, 7-dehydrocholesterol, lathosterol, desmosterol, and allocholesterol, partially restored by epicholesterol, and not restored by lanosterol, coprostanol, and 4-cholesten-3-one. These data support the hypothesis that the ability to form ordered domains is sufficient for a sterol to support ligand-induced activation of IR and IGF1R in intact mammalian cells.     

Negative regulation of the actin-regulating kinase Prk1p by patch localization-induced autophosphorylation

The Prk1 family of protein kinases are important regulators of endocytosis and actin cytoskeleton in some eukaryotic cells. In budding yeast, Prk1p phosphorylates numerous endocytic proteins including Pan1p and Sla1p. Prk1p has been observed to undergo autophosphorylation in vivo . In this study, we determined the sites and underlying role of the autophosphorylation. Two sites located in the noncatalytic region were identified to be the autophosphorylation sites. When the sites were mutated, the non-autophosphorylatable Prk1p phosphorylated Pan1p and Sla1p more efficiently than the wild-type kinase, suggesting a negative effect of the autophosphorylation. In addition, the dynamic properties of actin and the coat complex were also altered in the autophosphorylation mutant cells. Interestingly, the autophosphorylation of Prk1p was dependent on cortical localization of the kinase and could be induced by phosphorylated Sla1p. These results suggest that the autophosphorylation of Prk1p may represent a feedback mechanism possibly involved in fine-tuning the pace of progression during actin-coupled endocytosis.     

Hyperosmotic stress activates the insulin receptor in CHO cells

Stress factors, such as osmotic stress and genotoxic agents, activate stress kinases, whereas growth factors preferentially stimulate the structurally homologous mitogen-activated protein kinases, ERK1/2. Hyperosmolarity also has insulin-mimicking action as reflected by ERK1/2 activation and by the stimulation of glucose uptake in adipocytes. We examined to what extent hyperosmolarity activates components of the insulin receptor (IR) signalling pathway. CHO cells expressing the human IR were treated with 500 mM NaCl or 700 mM sorbitol and the activation of insulin signalling intermediates was studied. Hyperosmolarity induced tyrosine phosphorylation of the IR beta-subunit, and the adaptor proteins p52-Shc, p66-Shc, and IRS1. Furthermore, the stress kinases JNK and p38 were activated. When CHO cells were transfected with a kinase-dead IR (K1030R) mutant, hyperosmolarity did not induce tyrosine phosphorylation of the IR, indicating that hyperosmolarity induced IR autophosphorylation directly, rather than inducing phosphorylation by an exogenous tyrosine kinase. A partially purified and detergent-solubilized IR was not phosphorylated in response to hyperosmolarity, suggesting that hyperosmolarity activates the receptor only when present in the plasma membrane. In cells stably expressing the kinase-dead IR, IRS1 and Shc Tyr phosphorylation was abrogated, indicating that the hyperosmolarity signalling was dependent on an active IR tyrosine kinase. In contrast, the stress kinases p38 and JNK were normally activated by hyperosmolarity in the IR-K1030R mutant. We conclude that, at least in CHO cells, hyperosmolarity signals partially through IR autophosphorylation and subsequent activation of the IR downstream targets. This may be responsible for some of the insulin-mimicking effects of hyperosmolarity. The activation of stress kinases by hyperosmolarity occurs independent of the IR.     

Regulation of insulin receptor function by a small molecule insulin receptor activator

In type 2 diabetes mellitus, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. TLK19780, a non-peptide small molecule, is a new member of a novel class of anti-diabetic agents that function as activators of the insulin receptor (IR) beta-subunit tyrosine kinase. In HTC-IR cells, 20 microm TLK19780 enhanced maximal insulin-stimulated IR autophosphorylation 2-fold and increased insulin sensitivity 2-3-fold. In contrast, TLK19780 did not potentiate the action of insulin-like growth factor-1, indicating the selectivity of TLK19780 toward the IR. The predominant effect of TLK19780 was to increase the number of IR that underwent autophosphorylation. Kinetic studies indicated that TLK19780 acted very rapidly, with a maximal effect observed 2 min after addition to insulin-stimulated cells. In 3T3-L1 adipocytes, 5 microm TLK19780 enhanced insulin-stimulated glucose transport, increasing both the sensitivity and maximal responsiveness to insulin. These studies indicate that at low micromolar levels small IR activator molecules can enhance insulin action in various cultured cells and suggest that this effect is mediated by increasing the number of IR that are tyrosine-phosphorylated in response to insulin. These studies suggest that these types of molecules could be developed to treat type 2 diabetes and other clinical conditions associated with insulin resistance.     

Rho/ROCK/actin signaling regulates membrane androgen receptor induced apoptosis in prostate cancer cells

In this study we describe a novel Rho small GTPase dependent pathway that elicits apoptotic responses controlled by actin reorganization in hormone-sensitive LNCaP- and hormone insensitive DU145-prostate cancer cells stimulated with membrane androgen receptor selective agonists. Using an albumin-conjugated steroid, testosterone-BSA, we now show significant induction of actin polymerization and apoptosis that can be reversed by actin disrupting agents in both cell lines. Testosterone-BSA triggered RhoA/B and Cdc42 activation in DU145 cells followed by stimulation of downstream effectors ROCK, LIMK2 and ADF/destrin. Furthermore, dominant-negative RhoA, RhoB or Cdc42 mutants or pharmacological inhibitors of ROCK inhibited both actin organization and apoptosis in DU145 cells. Activation of RhoA/B and ROCK was also implicated in membrane androgen receptor-dependent actin polymerization and apoptosis in LNCaP cells. Our findings suggest that Rho small GTPases are major membrane androgen receptor effectors controlling actin reorganization and apoptosis in prostate cancer cells.     

Deficiency of IRTKS as an adaptor of insulin receptor leads to insulin resistance

IRTKS encodes a member of the IRSp53/MIM homology domain family, which has been shown to play an important role in the formation of plasma membrane protrusions. Although the phosphorylation of IRTKS occurs in response to insulin stimulation, the role of this protein in insulin signaling remains unknown. Here we show that IRTKS-deficient mice exhibit insulin resistance, including hyperglycemia, hyperinsulinemia, glucose intolerance, decreased insulin sensitivity, and increased hepatic glucose production. The administration of ectopic IRTKS can ameliorate the insulin resistance of IRTKS-deficient and diabetic mice. In parallel, the expression level of IRTKS was significantly decreased in diabetic mouse model. Furthermore, DNA hypermethylation of the IRTKS promoter was also observed in these subjects. We also show that IRTKS, as an adaptor of the insulin receptor (IR), modulates IR-IRS1-PI3K-AKT signaling via regulating the phosphorylation of IR. These findings add new insights into our understanding of insulin signaling and resistance.     

Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose

Insulin stimulates the autophosphorylation of tyrosine residues of the beta subunit of the insulin receptor (IR); this modified insulin-independent kinase has increased activity toward exogenous substrates in vitro. We show here that replacement of one or both of the twin tyrosines (residues 1162 and 1163) with phenylalanine results in a dramatic reduction in or loss of insulin-activated autophosphorylation and kinase activity in vitro. In vivo, these mutations not only result in a substantial decrease in insulin-stimulated IR autophosphorylation but also in a parallel decrease in the insulin-activated uptake of 2-deoxyglucose. Furthermore, a truncated IR protein (lacking the last 112 amino acids) has an unstable beta subunit; this mutant has no kinase activity in vitro or in vivo and does not mediate insulin-stimulated uptake of 2-deoxyglucose. IR autophosphorylation is thus implicated in the regulation of IR activities, with tyrosines 1162 and 1163 as major sites of this regulation.     

Generation of intracellular domain of insulin receptor tyrosine kinase by gamma-secretase

The proteolytic cleavage of a precursor protein into alpha- and beta-subunits by furin is required to form functional insulin receptor (IR). In this study, we examined if IR undergoes the additional presenilin (PS)/gamma-secretase-dependent processing. In cells treated with gamma-secretase inhibitors or expressing the dominant-negative PS1 variant led to the accumulation of an endogenous IR C-terminal fragment. In the presence of proteasome inhibitors, we detected a PS/gamma-secretase cleavage product of the IR, termed the IR intracellular domain (ICD). Cellular fractionation and confocal microscopy analyses showed that the IR-ICD is predominantly detected in the nucleus. These data indicate that IR is a tyrosine kinase receptor, which undergoes PS/gamma-secretase-dependent processing. We also show that the autophosphorylation levels of the IR beta-subunit upon insulin stimulation were decreased by the inactivation of PS/gamma-secretase, raising the possibility that the PS/gamma-secretase proteolysis of IR may play a modulatory role in insulin signaling.     

Spatial and temporal aspects and the interplay of Grb14 and protein tyrosine phosphatase-1B on the insulin receptor phosphorylation

Background

Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14 knockout studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling, in a tissue specific manner. Retinal cells are post-mitotic tissue, and insulin receptor (IR) activation is essential for retinal neuron survival. Retinal cells express protein tyrosine phosphatase-1B (PTP1B), which dephosphorylates IR and Grb14, a pseudosubstrate inhibitor of IR. This project asks the following major question: in retinal neurons, how does the IR overcome inactivation by PTP1B and Grb14?

Results

Our previous studies suggest that ablation of Grb14 results in decreased IR activation, due to increased PTP1B activity. Our research propounds that phosphorylation in the BPS region of Grb14 inhibits PTP1B activity, thereby promoting IR activation. We propose a model in which phosphorylation of the BPS region of Grb14 is the key element in promoting IR activation, and failure to undergo phosphorylation on Grb14 leads to both PTP1B and Grb14 exerting their negative roles in IR. Consistent with this hypothesis, we found decreased phosphorylation of Grb14 in diabetic type 1 Ins2^Akita mouse retinas. Decreased retinal IR activation has previously been reported in this mouse line.

Conclusions

Our results suggest that phosphorylation status of the BPS region of Grb14 determines the positive or negative role it will play in IR signaling.