Insulin-like growth factor-II is produced by, signals to and is an important survival factor for the mature podocyte in man and mouse.

Podocytes are crucial for preventing the passage of albumin into the urine and, when lost, are associated with the development of albuminuria, renal failure and cardiovascular disease. Podocytes have limited capacity to regenerate, therefore pro-survival mechanisms are critically important. Insulin-like growth factor-II (IGF-II) is a potent survival and growth factor; however, its major function is thought to be in prenatal development, when circulating levels are high. IGF-II has only previously been reported to continue to be expressed in discrete regions of the brain into adulthood in rodents, with systemic levels being undetectable. Using conditionally immortalized human and ex vivo adult mouse cells of the glomerulus, we demonstrated the podocyte to be the major glomerular source and target of IGF-II; it signals to this cell via the IGF-I receptor via the PI3 kinase and MAPK pathways. Functionally, a reduction in IGF signalling causes podocyte cell death in vitro and glomerular disease in vivo in an aged IGF-II transgenic mouse that produces approximately 60% of IGF-II due to a lack of the P2 promoter of this gene. Collectively, this work reveals the fundamental importance of IGF-II in the mature podocyte for glomerular health across mammalian species.

Insulin directly stimulates VEGF-A production in the glomerular podocyte.

Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.

High glucose causes dysfunction of the human glomerular endothelial glycocalyx.

The endothelial glycocalyx is a gel-like layer which covers the luminal side of blood vessels. The glomerular endothelial cell (GEnC) glycocalyx is composed of proteoglycan core proteins, glycosaminoglycan (GAG) chains, and sialoglycoproteins and has been shown to contribute to the selective sieving action of the glomerular capillary wall. Damage to the systemic endothelial glycocalyx has recently been associated with the onset of albuminuria in diabetics. In this study, we analyze the effects of high glucose on the biochemical structure of the GEnC glycocalyx and quantify functional changes in its protein-restrictive action. We used conditionally immortalized human GEnC. Proteoglycans were analyzed by Western blotting and indirect immunofluorescence. Biosynthesis of GAG was analyzed by radiolabeling and quantified by anion exchange chromatography. FITC-albumin was used to analyze macromolecular passage across GEnC monolayers using an established in vitro model. We observed a marked reduction in the biosynthesis of GAG by the GEnC under high-glucose conditions. Further analysis confirmed specific reduction in heparan sulfate GAG. Expression of proteoglycan core proteins remained unchanged. There was also a significant increase in the passage of albumin across GEnC monolayers under high-glucose conditions without affecting interendothelial junctions. These results reproduce changes in GEnC barrier properties caused by enzymatic removal of heparan sulfate from the GEnC glycocalyx. They provide direct evidence of high glucose-induced alterations in the GEnC glycocalyx and demonstrate changes to its function as a protein-restrictive layer, thus implicating glycocalyx damage in the pathogenesis of proteinuria in diabetes.

Rosiglitazone enhances glucose uptake in glomerular podocytes using the glucose transporter GLUT1.

AIMS/HYPOTHESIS: Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used increasingly in the treatment of type 2 diabetes. In the context of renal disease, PPARgamma agonists reduce microalbuminuria in diabetic nephropathy; however, the mechanisms underlying this effect are unknown. Glomerular podocytes are newly characterised insulin-sensitive cells and there is good evidence that they are targeted in diabetic nephropathy. In this study we investigated the functional and molecular effects of the PPARgamma agonist rosiglitazone on human podocytes. METHODS: Conditionally immortalised human podocytes were cultured with rosiglitazone and functional effects were measured with glucose-uptake assays. The effect of rosiglitazone on glucose uptake was also measured in 3T3-L1 adipocytes, nephrin-deficient podocytes, human glomerular endothelial cells, proximal tubular cells and podocytes treated with the NEFA palmitate. The role of the glucose transporter GLUT1 was investigated with immunofluorescence and small interfering RNA knockdown and the plasma membrane expression of GLUT1 was determined with bis-mannose photolabelling. RESULTS: Rosiglitazone significantly increased glucose uptake in wild-type podocytes and this was associated with translocation of GLUT1 to the plasma membrane. This effect was blocked with GLUT1 small interfering RNA. Nephrin-deficient podocytes, glomerular endothelial cells and proximal tubular cells did not increase glucose uptake in response to either insulin or rosiglitazone. Furthermore, rosiglitazone significantly increased basal and insulin-stimulated glucose uptake when podocytes were treated with the NEFA palmitate. CONCLUSIONS/INTERPRETATION: In conclusion, rosiglitazone has a direct and protective effect on glucose uptake in wild-type human podocytes. This represents a novel mechanism by which PPARgamma agonists may improve podocyte function in diabetic nephropathy.

GSK3: a SHAGGY frog story.

Glycogen synthase kinase 3 was discovered in mammals several years ago but only recently has it become clear that this enzyme is acutely regulated by hormones such as insulin and by growth factors. In mammals, it appears to be controlled by a signalling pathway linked to phosphoinositide 3-kinase and may regulate a range of biosynthetic processes. Evidence is now accumulating that GSK3 plays a key role in the regulation of cell fate and differentiation in many eukaryotic species.

Podocyte GSK3α is important for autophagy and its loss detrimental for glomerular function.

Podocytes are key cells in maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Glycogen synthase kinase 3 (GSK3) is a multi-functional serine/threonine kinase existing as two distinct but related isoforms (α and ß). In the podocyte it has previously been reported that inhibition of the ß isoform is beneficial in attenuating a variety of glomerular disease models but loss of both isoforms is catastrophic. However, it is not known what the role of GSK3α is in these cells. We now show that GSK3α is present and dynamically modulated in podocytes. When GSK3α is transgenically knocked down specifically in the podocytes of mice it causes mild but significant albuminuria by 6-weeks of life. Its loss also does not protect in models of diabetic or Adriamycin-induced nephropathy. In vitro deletion of podocyte GSK3α causes cell death and impaired autophagic flux suggesting it is important for this key cellular process. Collectively this work shows that GSK3α is important for podocyte health and that augmenting its function may be beneficial in treating glomerular disease.

Podocyte GSK3 is an evolutionarily conserved critical regulator of kidney function.

Albuminuria affects millions of people, and is an independent risk factor for kidney failure, cardiovascular morbidity and death. The key cell that prevents albuminuria is the terminally differentiated glomerular podocyte. Here we report the evolutionary importance of the enzyme Glycogen Synthase Kinase 3 (GSK3) for maintaining podocyte function in mice and the equivalent nephrocyte cell in Drosophila. Developmental deletion of both GSK3 isoforms (α and ß) in murine podocytes causes late neonatal death associated with massive albuminuria and renal failure. Similarly, silencing GSK3 in nephrocytes is developmentally lethal for this cell. Mature genetic or pharmacological podocyte/nephrocyte GSK3 inhibition is also detrimental; producing albuminuric kidney disease in mice and nephrocyte depletion in Drosophila. Mechanistically, GSK3 loss causes differentiated podocytes to re-enter the cell cycle and undergo mitotic catastrophe, modulated via the Hippo pathway but independent of Wnt-ß-catenin. This work clearly identifies GSK3 as a critical regulator of podocyte and hence kidney function.

Evidence for a role for protein kinase C in the stimulation of protein synthesis by insulin in swiss 3T3 fibroblasts.

We have examined the role of protein kinase C (PKC) in the stimulation of protein synthesis by insulin using two complementary approaches. In the first, fibroblasts were pretreated with phorbol esters to down-regulate PKC. In these cells, the effects of insulin and of phorbol esters on protein synthesis were completely abolished, although serum still elicited an effect approaching that seen in control cells. Secondly, we used newly developed inhibitors of PKC which, again, blocked the effects of insulin and phorbol esters without greatly reducing the response to serum. Thus PKC apparently plays an important role in the stimulation of translation by insulin.

Regulation of eukaryotic initiation factor eIF2B: glycogen synthase kinase-3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin.

Eukaryotic initiation factor eIF2B catalyses a key regulatory step in mRNA translation. eIF2B and total protein synthesis are acutely activated by insulin, and this requires phosphatidylinositol 3-kinase (PI 3-kinase). The epsilon-subunit of eIF2B is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is inactivated by insulin in a PI 3-kinase-dependent manner. Here we identify the phosphorylation site in eIF2Bepsilon as Ser540 and show that treatment of eIF2B with GSK-3 inhibits its activity. Ser540 is phosphorylated in intact cells and undergoes dephosphorylation in response to insulin. This is blocked by PI 3-kinase inhibitors. Insulin-induced dephosphorylation of this inhibitory site in eIF2B seems likely to be important in the overall activation of translation by this hormone.

Activation of translation initiation factor eIF2B by insulin requires phosphatidyl inositol 3-kinase.

Eukaryotic initiation factor eIF2B mediates a key regulatory step in peptide-chain initiation and is acutely activated by insulin, although, it is not clear how. Inhibitors of phosphatidylinositide 3-kinase blocked activation of eIF2B, although rapamycin, which inhibits the p70 S6 kinase pathway, did not. Furthermore, a dominant negative mutant of PI 3-kinase also prevented activation of eIF2B, while a Sos-mutant, which blocks MAP kinase activation, did not. The data demonstrate that a pathway distinct from MAP and p70 S6 kinases regulates eIF2B. Glycogen synthase kinase-3 (GSK-3) phosphorylates and inactivates eIF2B. In all cases, eIF2B and GSK-3 were regulated reciprocally. Dominant negative PI 3-kinase abolished the insulin-induced inhibition of GSK-3. These data strongly support the hypothesis that insulin activates eIF2B through a signalling pathway involving PI 3-kinase and inhibition of GSK-3.

Using green fluorescent protein to study intracellular signalling.

Subcellular compartmentalisation of signalling molecules is an important phenomenon not only in defining how a signalling pathway is activated but also in influencing the desired physiological output of that pathway (e.g. cell growth or differentiation, regulation of metabolism, cytoskeletal changes etc.). Biochemical analyses of protein and lipid compartmentalisation by, for example, subcellular fractionation presents many technical difficulties. However, this aspect of cell signalling research has seen a major revolution thanks to the cloning and availability of a variety of mutant green fluorescent protein derivatives with distinct molecular properties. Mutants with increased brightness, altered excitation and emission maxima, altered stability and differential sensitivity to pH, are now in widespread use for following the trafficking and function of proteins in living cells and for monitoring the intracellular environment. In this review we focus on some of the recent developments in the use of green fluorescent proteins for studying intracellular signalling pathways often with special reference to the actions of insulin. We also discuss the future utility of these proteins to analyse protein--protein interactions in signalling pathways using fluorescence resonance energy transfer.

VEGF and sVEGFR-1 in malignant pleural effusions: association with survival and pleurodesis outcomes.

VEGF is a key mediator of tumour growth and metastasis and is considered central to the formation of exudative pleural effusions. This study examined the relationship between levels of VEGF and its soluble receptor, sVEGFR-1 in the pleural fluid and plasma of patients with malignant pleural effusions and their association with pleurodesis outcomes and survival. 103 patients with malignant pleural effusions were recruited at their first presentation. Follow-up was to 6 months or death. Survival and pleurodesis outcomes were robustly ascertained. VEGF and sVEGFR-1 were measured in pleural fluid and plasma by ELISA. VEGF and sVEGFR-1 were present in significantly higher concentrations in pleural fluid than plasma. There was no significant correlation between mediators within or between sample types. There was no association between baseline pleural fluid VEGF or sVEGFR-1 levels and pleurodesis failure. In both sample types, survival was inversely associated with sVEGFR-1 and within the non-small cell lung cancer sub-group (n=26), a highly significant association between increased pleural fluid VEGF and sVEGFR-1 and reduced survival was demonstrated (p=0.02 and 0.004 respectively). In conclusion, we have shown for the first time that sVEGFR-1 can be reproducibly measured in pleural fluid from malignant effusions. High levels at presentation in those with non-small cell carcinoma are strongly associated with poor outcomes.

Regulation of small GTP-binding proteins by insulin.

Several members of the extensive family of small GTP-binding proteins are regulated by insulin, and have been implicated in insulin action on glucose uptake. These proteins are themselves negatively regulated by a series of specific GAPs (GTPase-activating proteins). Interestingly, there is increasing evidence to suggest that PKB (protein kinase B)-dependent phosphorylation of some GAPs may relieve this negative regulation and so lead to the activation of the target small GTP-binding protein. We review recent evidence that this may be the case, and place specific emphasis on the role of these pathways in insulin-stimulated glucose uptake.

Role of protein kinase B in insulin-regulated glucose uptake.

The activation of protein kinase B (or Akt) plays a central role in the stimulation of glucose uptake by insulin. Currently, however, numerous questions remain unanswered regarding the role of this kinase in bringing about this effect. For example, we do not know precisely where in the GLUT4 trafficking pathway this kinase acts. Nor do we know which protein substrates are responsible for mediating the effects of protein kinase B, although two recently identified proteins (AS160 and PIKfyve) may play a role. This paper addresses these important questions by reviewing recent progress in the field.

Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B.

We have studied the control of insulin-regulated protein kinases in Chinese hamster ovary cells transfected with the human insulin receptor (CHO.T cells). Among these enzymes is one that is obtained after chromatography of cell extracts on Mono-S, whose activity is decreased (7.3 +/- 1.9-fold) within 10 min of insulin treatment. This enzyme phosphorylates glycogen synthase and the largest subunit of protein synthesis eukaryotic initiation factor (eIF)-2B (the guanine nucleotide exchange factor). The kinase appears to be glycogen synthase kinase-3 (GSK-3), on the basis of: (1) its ability to phosphorylate a peptide based on the phosphorylation sites for GSK-3 in glycogen synthase, and (2) the finding that the fractions possessing this activity contain immunoreactive GSK-3, whose peak is coincident with that of kinase activity, as judged by immunoblotting using antibodies specific for the alpha- and beta-isoforms of GSK-3. The decrease in kinase activity induced by insulin was reversed by treatment of the column fractions with protein phosphatase-2A. These data indicate that insulin rapidly causes inactivation of GSK-3 and that this is due to phosphorylation of GSK-3. The implications of these findings for the control of glycogen and protein metabolism are discussed.

Regulation of protein synthesis in Swiss 3T3 fibroblasts. Rapid activation of the guanine-nucleotide-exchange factor by insulin and growth factors.

Insulin, whole serum, phorbol esters and epidermal growth factor each rapidly stimulate protein synthesis in serum-depleted Swiss 3T3 fibroblasts. The activation of protein synthesis by each of these agents is associated with stimulation of the activity of the guanine-nucleotide-exchange factor (GEF). This protein recycles the initiation factor eIF-2 by promoting exchange of GDP bound to eIF-2 for GTP. Activation of GEF is rapid, becoming maximal within 15 min. The degree of activation of GEF by these stimuli (to greater than 170% of control for insulin, serum or epidermal growth factor; 120% for phorbol dibutyrate) is more than enough to account for their effects on the overall rate of translation. Stimulation of protein synthesis and GEF activity occurs at low nanomolar insulin concentrations, indicating they are mediated through the insulin receptor. The best-characterized mechanism for regulating GEF activity is through changes in the phosphorylation of the smallest subunit of eIF-2 (eIF-2 alpha); however, none of the stimuli studied altered the level of phosphorylation of eIF-2 alpha in Swiss fibroblasts. It seems that direct regulation of GEF activity may be occurring here, and possible mechanisms for this are discussed.

Peptide substrates suitable for assaying glycogen synthase kinase-3 in crude cell extracts.

In this study we describe the characterization and use of new peptide substrates for assaying glycogen synthase kinase-3 (GSK-3) which are based on the sequence around the single GSK-3 phosphorylation site in the translation factor eIF2B. The new peptides offer important advantages over previous substrates, which were based on the sequence around the multiple GSK-3 phosphorylation sites in glycogen synthase (GS), for the assay of GSK-3 in cell extracts. In particular, decreases in GSK-3 activity following, e.g., insulin treatment, are partially or completely masked when the GS-based peptides are used but are readily measured using the new, eIF2B-based, peptides. The new peptides, unlike those based on GS, are therefore suitable for the assay of changes in GSK-3 activity in cell extracts without the need for prior immunoprecipitation or ion-exchange chromatography.

Phosphorylation of only serine-51 in protein synthesis initiation factor-2 is associated with inhibition of peptide-chain initiation in reticulocyte lysates.

We have examined the phosphorylation of the alpha-subunit of initiation factor-2 (eIF-2 alpha) in reticulocyte lysates in which translational shut-off was induced by haem-deficiency or by double-stranded RNA. To maximise the phosphorylation of eIF-2 alpha, lysates were supplemented with the broad spectrum phosphatase inhibitor microcystin. Under all conditions tested, serine-51 was the only residue to become labelled. This is consistent with the observation of only two species of eIF-2 alpha in isoelectric focusing/immunoblotting analyses of lysates treated as described above.

Identification of novel phosphorylation sites in the beta-subunit of translation initiation factor eIF-2.

Initiation factor eIF-2 (a trimer of subunits alpha, beta and gamma) attaches the initiator Met-tRNA to the ribosome during the initiation of translation in eukaryotic cells. Both the alpha and beta subunits can be phosphorylated although the sites in the beta-subunit have not previously been fully identified. Here we identify the sites at which eIF-2 beta is phosphorylated in vitro by three well-characterised protein kinases, casein kinase-2 (which phosphorylates serine residues-2 and -67), protein kinase C (serine-13) and cAMP-dependent protein kinase (serine-218). This constitutes an essential prerequisite for studying the phosphorylation of eIF-2 beta in vivo. Indeed, we present evidence that at least one of these sites (serine-67) is phosphorylated in reticulocytes. The major kinase activity against eIF-2 beta in reticulocyte lysates appears in CK-2 and protein phosphatase-2A is the principal enzyme responsible for dephosphorylation of eIF-2 beta phosphorylated by this kinase.

T-cell activation leads to rapid stimulation of translation initiation factor eIF2B and inactivation of glycogen synthase kinase-3.

Mitogenic stimulation of T-lymphocytes causes a rapid activation or protein synthesis, which reflects in part increased expression of many translation components. Their levels, however, rise more slowly than the rate of protein synthesis, indicating an enhancement of the efficiency of their utilization. Initiation factor eIF2B catalyzes a key regulatory step in the initiation of translation, and we have therefore studied its activity following T-cell activation. eIF2B activity rises quickly, increasing as early as 5 min after cell stimulation. This initial phase is followed by an additional slow but substantial increase in eIF2B activity. The level of eIF2B subunits did not change over the initial rapid phase but did increase at later time points. Northern analysis revealed that levels of eIF2B mRNA only rose during the later phase. The rapid activation of EIF2B following mitogenic stimulation of T-cells is therefore mediated by factors other than its own concentration. The largest (epsilon) subunit of eIF2B is a substrate for glycogen synthase kinase-3 (GSK-3), the activity of which rapidly decreases following T-cell activation. Since phosphorylation of eIF2B by GSK-3 appears to inhibit nucleotide exchange in vitro, this provides a potential mechanism by which eIF2B may be activated.