The role of cytoskeletal proteins in the mechanism of insulin release

This thesis is concerned with the role of /3-cell cytoskeletal proteins in the mechanism of insulin release from islets of experimental animals, the Aston obese diabetic hyperglycaemic (ob/ob) mouse and their lean littermates and the cultural insulin secreting /?-cell lines, HIT-TT5 and RINm5F. Investigations were carried out into the glucose induced insulin response of the lean and obese mouse islets and HIT-TI5 cells and the D-glyceraldehyde response of RINm5F cells using a static incubation system. Colchicine was found to inhibit insulin release from both lean and obese mouse islets more significantly than cultured TTT-TI5 and RINm5F cells. (Colchicine pre-treatment also inhibited the second phase of insulin release from perifused lean mouse islets and HIT-TI5 cells). Cytocha-lasin B, used to investigate the role of the microfilamentous system in the mechanism of insulin release enhanced insulin release from both lean and obese mouse islets to a significantly greater degree than that from cultured HIT-TI5 and RINm5F cells. Pre-treatment of isolated lean and obese mouse islets and cultured /?-cells with a combination of colchicine and cytochalasin B significantly reduced the insulin response of the HIT-TI5 and RINm5F cells compared with the control values suggesting that intact microtubules are more important for the sustained release of insulin than the microfilamentous system. However, the response was not so clearly defined with the lean and obese mouse islets. Tubulin was separated from the extracts of lean mouse islets and the HIT-TI5 and RINm5F cells and actin was separated from all of the cell types including the obese mouse islets by SDS- polyacrylamide electrophoresis. A tubulin radioimmunoassay and a colchicine binding assay were developed to measure the tubulin content of lean and obese mouse islets, and the shift between the proportions of tubulin dimers and polymerized tubulin under stimulatory and non-stimulatory conditions. The assay methods developed were not prone to be accurate, sensitive and precise but gave some indication of the shift from unpolymerised to polymerised tubulin during glucose stimulated insulin release. These studies show that microtubules do play a fundamental role in the mechanism of insulin release from both islets and cultured HIT-TI5 and RINm5F cells.

Insulin release from cultured B-cells

Established RlNm5F and lN111 R1 and newly available HlT-T15 and UMR 407/3 B-cell lines have been successfully maintained in vitro. With the exclusion of UMR 407/3 cells, all lines were continuously propagable. Doubling times and plating efficiencies for HlT-T15, RlNm5F, lN111 R1 and UMR 407/3 cells were 20 hours and 85%, 31 hours and 76%, 24 hours and 80% and 38 hours and 94% respectively. All the cell lines were anchorage dependent, but only UMR 407/3 cells grew to confluence. Only HlT-T15 and UMR 407/3 cells produced a true insulin response to glucose but glucose markedly increased the rate of D-[U14C]glucose oxidation by all the cell lines. Glucose induced insulin release from HlT-T15 cells was biphasic with an exaggerated first phase. Insulin release from HlT-T15, RlNm5F and IN111 R1 cells was stimulated by amino acids and sulphonylureas. Glucagon stimulated insulin release from HlT-T15 and RlNm5F cells while somatostatin and pancreatic polypeptide inhibited release. These observations suggest that net insulin release from the whole islet may be the result of significant paracrine interaction. HlT-T15 and RlNm5F cell insulin release was stimulated by forskolin and inhibited by imidazole. Ca2+ channel blockade and calmodulin inhibition suppressed insulin release from HlT-T15, RlNm5F and IN111 R1 cells. In addition phorbol esters stimulated insulin release from RlNm5F cells. These data implicate cAMP, Ca2+ and protein kinase-C in the regulation of insulin release from cultured B-cells. Acetylcholine increased insulin release from HlT-T15 and RlNm5F cells. Inhibition of the response by atropine confirmed the involvement of muscarinic receptors. HlT-T15 cell insulin release was also inhibited by adrenaline. These observations suggest a possible role for the autonomic nervous system in the modulation of insulin release. Preliminary studies with a human insulinoma maintained in monolayer culture have demonstrated a limited life span of some seven weeks, a continuous low level of insulin release but no insulin response to glucose challenge.

Insulin gene therapy for the treatment of diabetes mellitus

Currently available treatments for insulin-dependent diabetes mellitus are often inadequate in terms of both efficacy and patient compliance. Gene therapy offers the possibility of a novel and improved method by which exogenous insulin can be delivered to a patient. This was approached in the present study by constructing a novel insulin-secreting cell line. For the purposes of this work immortalized cell lines were used. Fibroblasts and pituitary cells were transfected with the human preproisinulin gene to create stable lines of proinsulin- and insulin-secreting cells. The effect of known β-cell secretagogues on these cells were investigated, and found mostly to have no stimulatory effect, although IBMX, arginine and ZnSO4 each increased the rate of secretion. Cyclosporin (CyA) is currently the immunosuppresant of choice for transplant recipients; the effect of this treatment on endogenous β-cell function was assessed both in vivo and in vitro. Therapeutic doses of CyA were found to reduce plasma insulin concentrations and to impair glucose tolerance. The effect of immunoisolation on insulin release by HIT T15 cells was also investigated. The presence of an alginate membrane was found to severely impair insulin release. For the first implantation of the insulin-secreting cells, the animal model selected was the athymic nude mouse. This animal is immunoincompetent, and hence the use of an immunosuppressive regimen is circumvented. Graft function was assessed by measurement of plasma human C peptide concentrations, using a highly specific assay. Intraperitoneal implantation of genetically manipulated insulin-secreting pituitary cells into nude mice subsequently treated with a large dose of streptozotocin (STZ) resulted in a significantly delayed onset of hyperglycaemia when compared to control animals. Consumption of a ZnSO4 solution was shown to increase human C peptide release by the implant. Ensuing studies in nude mice examined the efficacy of different implantation sites, and included histochemical examination of the tumours. Aldehyde fuchsin staining and immunocytochemical processing demonstrated the presence of insulin containing cells within the excised tissue. Following initial investigations in nude mice, implantation studies were performed in CyA-immunosuppressed normal and STZ-diabetic mice. Graft function was found to be less efficacious, possibly due to the subcutaneous implantation site, or to the immunosuppresive regimen. Histochemical and transmission electron microscopic analysis of the tumour-like cell clusters found at autopsy revealed necrosis of cells at the core, but essentially normal cell morphology, with dense secretory granules in peripheral cells. The thesis provides evidence that gene therapy offers a feasibly new approach to insulin delivery.

Effects of metformin on BRIN-BD11 beta-cell insulin secretory desensitization induced by prolonged exposure to sulphonylureas

Aims: Prolonged exposure of pancreatic beta-cells in vitro to the sulphonylureas tolbutamide and glibenclamide induces subsequent desensitization of insulinotropic pathways. Clinically, the insulin-sensitizing biguanide drug metformin is often administered alongside sulphonylurea as antidiabetic therapy. The present study examines the functional effects of metformin (200 µM) on tolbutamide- and glibenclamide-induced desensitisation. Methods: Acute and prolonged (18 h) effects of exposure to tolbutamide and glibenclamide alone, or in the presence of metformin, were examined in insulin-secreting BRIN-BD11 cells. Results: In acute 20 min incubations at 1.1 mM glucose, metformin increased (1.2-1.7-fold; p <0.001) the insulin-releasing actions of tolbutamide and glibenclamide. At 16.7 mM glucose, metformin significantly enhanced glibenclamide-induced insulin release at all concentrations (50-400 µM) examined, but tolbutamide-stimulated insulin secretion was only augmented at higher concentrations (300-400 µM). Exposure for 18 h to 100 µM tolbutamide or glibenclamide significantly impaired insulin release in response to glucose and a broad range of insulin secretagogues. Concomitant culture with metformin (200 µM) prevented or partially reversed many of the adverse effects on K channel dependent and independent insulinotropic pathways. Beneficial effects of metformin were also observed in cells exposed to glibenclamide for 18 h with significant improvements in the insulin secretory responsiveness to alanine, GLP-1 and sulphonylureas. The decrease of viable cell numbers observed with glibenclamide was reversed by co-culture with metformin, but cellular insulin content was depressed. Conclusions: The results suggest that metformin can prevent the aspects of sulphonylurea-induced beta-cell desensitization. © 2010 Blackwell Publishing Ltd.

Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.

Control of the endocrine pancreas by gastrointestinal peptides

This thesis concerns the mechanism through which enteral delivery of glucose results in a larger insulin response than an equivalent parenteral glucose load. Preliminary studies in which mice received a glucose solution either intragastrically or intraperitoneally confirmed this phenomenon. An important regulatory system in this respect is the entero-insular axis, through which insulin secretion is influenced by neural and endocrine communication between the gastrointestinal tract and the pancreatic islets of Langerhans. Using an in vitro system involving static incubation of isolated (by collagenase digestion) islets of Langerhans, the effect of a variety of gastrointestinal peptides on the secretion of the four main islet hormones, namely insulin, glucagon, somatostatin and pancreatic polypeptide, was studied. The gastrointestinal peptides investigated in this study were the secretin family, comprising secretin, glucagon, gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and growth hormone releasing factor (GRF). Gastrin releasing peptide (GRP) was also studied. The results showed that insulin release was stimulated by all peptides studied except PHI, glucagon release was stimulated by all peptides tested, except GRF which suppressed glucagon release, somatostatin release was stimulated by GIP and GRF but suppressed by VIP, PHI, glucagon and secretin, and PP release was stimulated by GIP and GRF, but suppressed by PHI. The insulinotropic effect of GRP was investigated further. A perifusion system was used to examine the time-course of insulin release from isolated islets after stimulation with GRP. GRP was shown to be insulinotropic only in the presence of physiologically elevated glucose concentrations and both first and second phases of insulin release were augmented. There was no effect at substimulatory or very high glucose concentrations. Studies using a cultured insulin-secreting islet cell line, the RINm5F cell line, were undertaken to elucidate the intracellular mechanism of action of GRP. This peptide did not enhance insulin release via an augmentation of glucose metabolism, or via the adenylate cyclase/cyclic AMP secondary messenger system. The pattern of changes of cytosolic free calcium in response to GRP, which involved both mobilization of intracellular stores and an influx of extracellular calcium, suggested the involvement of phosphatidylinositol bisphosphate breakdown as a mediator of the effect of GRP on insulin secretion.

Microencapsulation strategies for islet transplantation

A variety of islet microencapsulation techniques have been investigated to establish which method provides the least occlusive barrier to net insulin release in vitro, and optimum biocompatibility for islet implantation in vivo. NMRI mouse islets have been microencapsulated with Na+ -alginate-poly-L-lysine (PLL)/poly-L-ornithine (PLO)-alginate, Ba2+ -alginate and agarose gels. Both free and microencapsulated islets responded to glucose challenge in static incubation and perifusion by significantly increasing their rate of insulin release and theophylline significantly potentiated the insulin response to glucose. While little insulin was released from microencapsulated islets after short term (2 hours) static incubation, significantly greater amounts were released in response to glucose challenge after extended (8-24 hours) incubation. However, insulin release from all types of microencapsulated islets was significantly reduced compared with free islets. Na+ -alginate-PLO-alginate microencapsulated islets were significantly more responsive to elevated glucose than Na+ -alginate-PLL-alginate microencapsulated islets, due to the enhanced porosity of PLO membranes. The outer alginate layer created a significant barrier to glucose/insulin exchange and reduced the insulin responsiveness of microencapsulated islets to glucose. Ba2+ -alginate membrane coated islets, generated by the density gradient method, were the most responsive to glucose challenge. Low concentrations of NG-monomethyl L-arginine (L-NMMA) had no significant effect on glucose stimulated insulin release from either free or microencapsulated islets. However, 1.0 mmol/1 L-NMMA significantly inhibited the insulin response of both free and microencapsulated islets to glucose challenge. In vivo work designed to evaluate the extent of pericapsular fibrosis after 28 days ip. and sc. implantation of microencapsulated islets into STZ-diabetic recipients, revealed that the inclusion of islets within microcapsules increased their immunogenicity and markedly increased the extent of pericapsular fibrosis. When the outer alginate layer was omitted from microcapsules, little or no pericapsular mononuclear cell deposition was observed. The subcutaneous site was not suitable for microencapsulated islet transplantation in NMRI recipient mice. Systemic immunosuppression using cyclosporin A was effective in preventing pericapsular mononuclear cell deposition, while L-NMMA loading into microcapsules had no significant effect on pericapsular fibrosis, although it did maintain the integrity of microencapsulated islets.

Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets

Elevated islet uncoupling protein-2 (UCP-2) impairs β-cell function and UCP-2 may be increased in clinical obesity and diabetes. We investigated the effects of glucose and leptin on UCP-2 expression in isolated human islets. Human islets were incubated for 24 h with glucose (5.5–22 mmol/l)±leptin (0–10 nmol/l). Some islet batches were incubated at high (22 mmol/l), and subsequently lower (5.5 mmol/l), glucose to assess reversibility of effects. Leptin effects on insulin release were also measured. Glucose dose-dependently increased UCP-2 expression in all islet batches, maximally by three-fold. This was not fully reversed by subsequently reduced glucose levels. Leptin decreased UCP-2 expression by up to 75%, and maximally inhibited insulin release by 47%, at 22 mmol/l glucose. This is the first report of UCP-2 expression in human islets and provides novel evidence of its role in the loss of β-cell function in diabetes.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala8-substituted analogues of GLP-1, (Abu8)GLP-1 and (Val8)GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu8)GLP-1 and (Val8)GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu8)GLP-1 and (Val8)GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val8)GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu8 )GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val8)GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala8 in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val8)GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Cachexia in MAC16 adenocarcinoma:Suppression of hunger despite normal regulation of leptin, insulin and hypothalamic neuropeptide Y

Weight loss normally stimulates hunger, through mechanisms that include falls in circulating leptin and insulin, leading to stimulation of hypothalamic neuropeptide Y (NPY). Here, we investigated the leptin, insulin and NPY to clarify why hunger is suppressed in mice with severe cachexia due to the MAC16 adenocarcinoma. MAC16-bearing mice progressively lost weight (19% below controls) and fat (-61%) over 16 days after tumour transplantation, while total food intake fell by 10%. Pair-fed mice showed less wasting, with final weight being 9% and fat mass 25% below controls. Plasma leptin fell by 85% in MAC16 and 51% in pair-fed mice, in proportion to loss of fat. Plasma insulin was also reduced by 49% in MAC16 and 53% in pair-fed groups. Hypothalamic leptin receptor (OB-Rb) mRNA was significantly increased in both MAC16 (+223%) and pair-fed (+192%) mice. Hypothalamic NPY mRNA was also significantly raised in MAC16 (+152%) and pair-fed (+99%) groups, showing negative correlations with plasma leptin and insulin, and a positive association with OB-Rb mRNA. In MAC16-induced cachexia, leptin production and hypothalamic OB-Rb and NPY expression are regulated appropriately in response to fat depletion. Therefore, suppression of hunger is probably due to tumour products that inhibit NPY transport or release, or that interfere with neuronal targets downstream of NPY.

The controlled release of macromolecules from macroporous hydrophyllic polymer matrices

This work has used novel polymer design and fabrication technology to generate bead form polymer based systems, with variable, yet controlled release properties, specifically for the delivery of macromolecules, essentially peptides of therapeutic interest. The work involved investigation of the potential interaction between matrix ultrastructural morphology, in vitro release kinetics, bioactivity and immunoreactivity of selected macromolecules with limited hydrolytic stability, delivered from controlled release vehicles. The underlying principle involved photo-polymerisation of the monomer, hydroxyethyl methacrylate, around frozen ice crystals, leading to the production of a macroporous hydrophilic matrix. Bead form matrices were fabricated in controllable size ranges in the region of 100µm - 3mm in diameter. The initial stages of the project involved the study of how variables, delivery speed of the monomer and stirring speed of the non solvent, affectedthe formation of macroporous bead form matrices. From this an optimal bench system for bead production was developed. Careful selection of monomer, solvents, crosslinking agent and polymerisation conditions led to a variable but controllable distribution of pore sizes (0.5 - 4µm). Release of surrogate macromolecules, bovine serum albumin and FITC-linked dextrans, enabled factors relating to the size and solubility of the macromolecule on the rate of release to be studied. Incorporation of bioactive macromolecules allowed retained bioactivity to be determined (glucose oxidase and interleukin-2), whilst the release of insulin enabled determination of both bioactivity (using rat epididymal fat pad) and immunoreactivity (RIA). The work carried out has led to the generation of macroporous bead form matrices, fabricated from a tissue biocompatible hydrogel, capable of the sustained, controlled release of biologically active peptides, with potential use in the pharmaceutical and agrochemical industries.

Loss of Akt activity increases circulating soluble endoglin release in preeclampsia:identification of inter-dependency between Akt-1 and heme oxygenase-1

Aims - Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-ß (TGF-ß) signalling, which is known to be elevated in preeclampsia. Methods and results - Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. Conclusion - The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction.

Reduced postprandial proinsulinaemia and 32-33 split proinsulinaemia after a mixed meal in type 2 diabetic patients following sensitization to insulin with pioglitazone

Objective: Reduced insulin sensitivity associated with fasting hyperproinsulinaemia is common in type 2 diabetes. Proinsulinaemia is an established independent cardiovascular risk factor. The objective was to investigate fasting and postprandial release of insulin, proinsulin (PI) and 32-33 split proinsulin (SPI) before and after sensitization to insulin with pioglitazone compared to a group treated with glibenclamide. Design and patients: A randomized double-blind placebo-controlled trial. Twenty-two type 2 diabetic patients were recruited along with 10 normal subjects. After 4 weeks washout, patients received a mixed meal and were assigned to receive pioglitazone or glibenclamide for 20 weeks, after which patients received another identical test meal. The treatment regimes were designed to maintain glycaemic control (HbA1c) at pretreatment levels so that ß-cells received an equivalent glycaemic stimulus for both test meals. Measurements: Plasma insulin, PI, SPI and glucose concentrations were measured over an 8-h postprandial period. The output of PI and SPI was measured as the integrated postprandial response (area under the curve, AUC). Results: Pioglitazone treatment resulted in a significant reduction in fasting levels of PI and SPI compared to those of the controls. Postprandially, pioglitazone treatment had no effect on the insulin AUC response to the meal but significantly reduced the PI and SPI AUCs. Glibenclamide increased fasting insulin and the postprandial insulin AUC but had no effect on the PI and SPI AUCs. Conclusions: Sensitization to insulin with pioglitazone reduces the amount of insulin precursor species present in fasting and postprandially and may reduce cardiovascular risk. © 2007 The Authors.

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Cα (PKCα), degradation of inhibitor-κB (I-κB) and nuclear migration of nuclear factor-κB (NF-κB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the α-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCα by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 μM), an inhibitor of eIF2α dephosphorylation, as was activation of PKCα. In addition myotubes transfected with a dominant-negative PKR (PKRΔ6) showed no release of arachidonate in response to Ang II, and no activation of PKCα. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA2/PKC pathway leading to activation of NF-κB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR. © 2007 Elsevier Inc. All rights reserved.