Complexation of novel thiomers and insulin to protect against in vitro enzymatic degradation - towards oral insulin delivery.

A significant barrier to oral insulin delivery is its enzymatic degradation in the gut. Nano-sized polymer-insulin polyelectrolyte complexes (PECS) have been developed to protect insulin against enzymatic degradation. Poly(allylamine) (Paa) was trimethylated to yield QPaa. Thiolation of Paa and QPaa was achieved by attaching either N-acetylcysteine (NAC) or thiobutylamidine (TBA) ligands (Paa-NAC/QPaa-NAC and Paa-TBA/QPaa-TBA thiomers). PEC formulations were prepared in Tris buffer (pH 7.4) at various polymer: insulin mass ratios (0.2:1-2:1). PECS were characterized by %transmittance of light and photon correlation spectroscopy. Insulin complexation efficiency and enzyme-protective effect of these complexes were determined by HPLC. Complexation with insulin was found to be optimal at mass ratios of 0.4-1:1 for all polymers. PECS in this mass range were positively-charged (20-40 mV), nanoparticles (50-200 nm), with high insulin complexation efficiency (>90%). Complexation with TBA polymers appeared to result in disulfide bridge formation between the polymers and insulin. In vitro enzymatic degradation assays of QPaa, Paa-NAC, and QPaa-NAC PECS showed that they all offered some protection against insulin degradation by trypsin and α-chymotrypsin, but not from pepsin. QPaa-NAC complexes with insulin are the most promising formulation for future work, given their ability to offer protection against intestinal enzymes. This work highlights the importance of optimizing polymer structure in the delivery of proteins.

Insulin and insulin-like growth factor-I responsiveness and signalling mechanisms in C2C12 satellite cells: effect of differentiation and fusion.

In proliferating C2C12 myoblasts, serum and physiological concentrations of insulin and IGF-I stimulated protein synthesis and RNA accretion. After fusion, the multinucleated myotubes remained responsive to serum but not to insulin or IGF-I, even though both insulin and type-I IGF receptor mRNAs increased in abundance. Protein synthetic responses to insulin and IGF-I in myoblasts were not inhibited by dexamethasone, ibuprofen or Ro-31-8220, thus phospholipase A2, cyclo-oxygenase and protein kinase C did not appear to be involved in the signalling mechanisms. Neither apparently were polyphosphoinositide-specific phospholipase C or phospholipase D since neither hormone increased inositol phosphate, phosphatidic acid, choline or phosphatidylbutanol production. Only the phosphatidylinositol-3-kinase inhibitor, wortmannin, and the 70 kDa S6-kinase inhibitor, rapamycin, wholly or partially blocked the effects of insulin and IGF-I on protein synthesis. 2-deoxyglucose uptake remained responsive to insulin and IGF-I after fusion and was also inhibited by wortmannin. The results suggest that the loss of responsiveness after fusion is not due to loss of receptors, but to the uncoupling of a post-receptor pathway, occurring after the divergence of the glucose transport and protein synthesis signalling systems, and that, if wortmannin acts at a single site, this is prior to that point of divergence.

Insulin receptor and glucose transporter mRNA expression in skeletal muscle of genetically obese Zucker rats.

Insulin resistance and defective glucose transport are associated with muscle tissue in the genetically obese Zucker rat and are accompanied by changes in the number of insulin receptors and the availability of glucose transporters. The present study was carried out to assess whether, in male Zucker rats at 10 weeks of age, these defects were reflected by changes in the levels of mRNAs for the insulin receptor and for the insulin responsive glucose transporter (GLUT-4). Total RNA was extracted from plantaris and soleus muscles and the levels of insulin receptor and GLUT-4 mRNAs and 18 S rRNA were determined by Northern hybridization and quantified by image analysis of the autoradiographs. A 50% increase in the level of insulin receptor mRNA was detected in both the plantaris and the soleus muscle from the obese rats compared to the lean rats. No change in the level of GLUT-4 mRNA was detected in the plantaris muscle although increases were observed in the soleus muscle from the obese rats.

High glucose concentration decreases insulin-like growth factor type 1-mediated mitogen-activated protein kinase activation in bovine retinal endothelial cells.

Clinical trials have incontrovertibly demonstrated that the onset and progression of diabetic retinopathy (DR) is influenced by the control of glucose levels in patients. In the present study, we examined the effect of glucose concentration on the responsiveness of bovine retinal endothelial cells (BREC) to insulin-like growth factor type 1 (IGF-1). Retinal endothelial cells were isolated from bovine retina and cultured in 5 or 20 mmol/L glucose with or without 100 ng/mL IGF-1. The level of cell growth and p42/44 and p38 mitogen-activated protein kinase (MAPK) activation was determined using the alamarBlue (Serotech) assay and Western blotting, respectively. IGF-1 significantly enhanced cell growth in BREC exposed to 5 mmol/L glucose but not in cells exposed to high glucose concentrations (20 mmol/L). IGF-1 induced a transient activation of p42/44 MAPK, with peak activation at 15 minutes in cells exposed to 5 mmol/L glucose; however, no increase in p42/44 MAPK was evident at the higher glucose concentration of 20 mmol/L. There was no significant change in the level of p38 MAPK during the time period examined when IGF-1 was also present. However, high glucose concentrations alone increased the level of p38 MAPK after 60 minutes and the level of p42/44 MAPK after only 15 minutes exposure in 20 mmol/L glucose. Thus, BREC exposed to high glucose concentrations are not sensitive to IGF-1 and this is due, at least in part, to a reduced activation of the p42/44 MAPK pathway. Furthermore, the presence of IGF-1 appears to exert a protective effect on the cells in high glucose concentration by preventing progression through the cell cycle.

Regulation of transforming growth factor-beta, basic fibroblast growth factor, and vascular endothelial cell growth factor mRNA in peripheral blood leukocytes in patients with diabetic retinopathy.

In the present study, we examined the effect of glucose concentration on the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-beta) mRNA using reverse transcriptase-polymerase chain reaction (RT-betaCR) in normal healthy leukocytes in vitro and in leukocytes from patients with type 1 diabetes mellitus. In vitro, the level of TGF-beta mRNA was altered in response to the glucose concentration (maximum at 10 mmol/L), while bFGF mRNA remained relatively constant and VEGF mRNA varied with no clear correlation with the glucose concentration. Leukocytes from type 1 patients showed no difference in bFGF or TGF-beta mRNA levels compared with age-matched healthy controls. However, VEGF mRNA was significantly lower in type 1 patients compared with controls (P < .05). When the patients were subtyped according to the severity of retinopathy, the level of TGF-beta mRNA was elevated selectively in patients with evidence of active new retinal vessels (P < .01) and VEGF121 mRNA was reduced in patients with mild to moderate retinopathy. Thus, leukocyte growth factor mRNAs respond to acute changes in the glucose concentration in vitro, and are differentially expressed in type 1 diabetic patients during the course of the disease.

Altered L-selectin expression in lymphocytes and increased adhesion to endothelium in patients with diabetic retinopathy.

AIM: To investigate L-selectin expression and shedding in patients with and without retinopathy and to determine if any observed changes are reflected by a functional change in the adhesion of leucocytes to an endothelial monolayer. METHODS: Age matched diabetic patients (26 with retinopathy, 19 without retinopathy) were compared to 24 non-diabetic controls to determine L-selectin surface protein expression, L-selectin mRNA production, and serum L-selectin levels by flow cytometry, RT-PCR, and ELISA, respectively. An adhesion assay was used to determine the binding of lymphocytes from the respective test groups to a monolayer of human endothelial cells. RESULTS: Significantly reduced (p = 0.004) L-selectin expression was demonstrated on lymphocytes (CD3+) from patients with diabetes compared to controls, the lowest levels being found in those with diabetic retinopathy (p = 0.004). L-selectin mRNA levels (p = 0.007) were significantly higher in the retinopathy group than in the no retinopathy group. Serum L-selectin levels were significantly higher (p = 0.04) in those with retinopathy compared to controls. Lymphocyte adhesion relative to control (100%) was essentially unchanged (84.0% (SD 27.7%), p = 0.15) for diabetic patients with no retinopathy and was markedly increased (192% (37.6%)) for those with retinopathy (p = 0.0001). CONCLUSION: Lymphocyte activation, reduced surface L-selectin, increased circulating L-selectin, and a corresponding increase in adhesion of patients' cells using an in vitro assay, is evident in people with diabetic retinopathy. This suggests a role for lymphocyte activation in the pathogenesis of diabetic retinopathy.

Regulation of plasminogen activation by TGF-beta in cultured human retinal endothelial cells.

BACKGROUND/AIMS: Regulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor beta (TGF-beta) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-beta in retinal angiogenesis in the context of diabetes mellitus. METHODS: Human retinal endothelial cells (HREC) were isolated from donor eyes and used between passages 4-8. The cells were cultured in medium supplemented with 2, 5, 15, or 25 mM glucose, plus or minus TGF-beta (1 ng/ml). The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor type 1 (PAI-1) in cell conditioned medium were determined by ELISA and the level of PAI-1 mRNA was determined using northern hybridisation. Cell associated plasminogen activity was determined using a clot lysis assay and a chromogenic assay. RESULTS: Under basal conditions (5 mM glucose), HREC produced PAI-1, t-PA, and trace amounts of u-PA. Cell surface plasminogen activation observed by lysis of fibrin or by cleavage of chromogenic substrate, was mediated by t-PA. Glucose at varying concentrations (2-25 mM) had no significant effect on t-PA mediated clot lysis. In contrast, treatment with TGF-beta resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-beta did not occur uniformly, the 2.3 kb mRNA transcript was preferentially increased in comparison with the 3.2 kb mRNA (p<0.05). CONCLUSIONS: These data demonstrate that TGF-beta increases PAI-1 and decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.

The persistence of infectious pancreatic necrosis virus in Atlantic salmon.

Atlantic salmon leucocytes from Infectious Pancreatic Necrosis Virus (IPNV) carriers showed a suppressed response to phytohemagglutinin stimulation compared with uninfected controls. A significant degree of inhibition of DNA synthesis was observed using 3H-thymidine incorporation. IPNV was isolated from 41% of the stimulated leucocyte cultures supernatants, while only 6% of the unstimulated cultures were found to be positive.

Glucose-dependent regulation of DNA synthesis in bovine retinal endothelial cells.

PURPOSE: Clinical trials have demonstrated that the onset and progression of diabetic retinopathy is influenced by the glucose control of the patient. The disease is characterised by the coexistence of impaired cell growth and excessive cell proliferation, and we wished to determine the effect that glucose has upon these parameters. METHODS: Bovine retinal endothelial cells were exposed to a range of glucose concentrations from 0-25 mmol/l. The level of DNA synthesis and cell number was then determined using pulse labelling with tritiated thymidine and a Coomassie blue dye-based assay, respectively. RESULTS: The level of DNA synthesis declined significantly as the concentration of glucose increased. DNA synthesis was further decreased by the presence of an inhibitor of PI3 kinase (Wortmannin). The decline in DNA synthesis was abrogated by the presence of a protein kinase C (PKC) inhibitor or by incubating the cells with antibodies specific for the GLUT-1 and GLUT-3 specific isoforms of glucose transporter proteins. TGF-beta antibody significantly increased the level of DNA synthesis in cells exposed to high concentrations of glucose. The changes that are observed in the level of DNA synthesis was not coincident with any significant changes in cell number as measured by the Coomassie blue assay. CONCLUSIONS: This demonstrated that the decline in DNA synthesis is dependent upon the entry of glucose into the cells and that this is mediated via a PKC dependent pathway.

Glucose-mediated regulation of transforming growth factor-beta (TGF-beta) and TGF-beta receptors in human retinal endothelial cells.

PURPOSE: Diabetic retinopathy is a micro-angiopathy affecting predominantly small vessels of the retina. Clinical trials have demonstrated a strong association between tight glucose control and a reduction in the incidence and the severity of diabetic retinopathy. Transforming growth factor beta (TGF-beta) is involved in the control of endothelial cell proliferation, adhesion, and deposition of extracellular matrix, thus TGF-beta may play a role in the control of endothelial cell proliferation seen in the disease. We wished to investigate the regulation of transforming growth factor beta and its receptors (type I and II) in human retinal endothelial cells exposed to a range of glucose concentrations. METHODS: Human retinal endothelial cells were isolated from donor eyes, cultured in vitro and exposed to a range of glucose concentrations (0-25 mmol/l). TGF-beta protein and mRNA levels were determined by ELISA and Northern analysis, respectively. The binding affinities and TGF-beta receptor numbers were defined using a binding assay. RESULTS: Northern hybridisation and ELISA showed that after 8 hours, the level of TGF-beta mRNA and protein was significantly higher at 15mmol/l compared to 5, 20 or 25mmol/ l. Binding assays showed that for high glucose (25 mmol/l), human retinal endothelial cells express a population of TGF-beta receptors with higher affinity for its ligand than at 5 or 15 mmol/l. CONCLUSIONS: These results demonstrate that glucose regulates TGF-beta mRNA and protein production and also TGF-beta receptor expression in human retinal endothelial cells. Thus, the glucose-mediated changes that occur in diabetic patients may expose human retinal endothelial cells to potential angiogenic factors which may influence disease progression.

Triploidy induction in recently fertilized Atlantic salmon ova using anaesthetics.

The comparative effectiveness of five general anaesthetics as agents for inducing triploidy in recently fertilized Atlantic salmon ova is reported. Triploid rates and triploid yields following the exposures of eggs from a single female fish to nitrous oxide, and separately to Freon 22, were shown to be related to the partial pressure and duration of exposure. The maximum triploid yield (79.7%) was observed following exposure to nitrous oxide at 11 atmospheres (atm) for the first 0 min after fertilization. Triploid yields following exposure to Freon (at 1 and 3 atm) were generally lower, the highest value (43.7%) occurring after exposure at ambient pressure for the first hour after fertilization. Triploid yields in these same eggs were low ( < 10%) when halothane and ethrane were used at partial pressures in the range 0.05-0.2 atm and cyclopropane at atmospheric pressure was ineffective. In a subsequent season the eggs from ten different individual female Atlantic salmon were shown to vary little in susceptibility following exposure to nitrous oxide treatment (0-30 min after fertilization, 11 atm) and experienced high yields of triploids (mean=80.4 +/- 6.6% SD).

Regulation of glucose transporter (GLUT 3) and aldose reductase mRNA inbovine retinal endothelial cells and retinal pericytes in high glucose and high galactose culture.

The regulation of GLUT-3 and aldose reductase mRNA in retinal endothelial cells and retinal pericytes was studied in response to variations in the extracellular concentration of hexoses. In physiological concentrations of glucose (5 mmol/l), an increase in the level of GLUT-3 mRNA was observed in cultured cells compared to the level of mRNA found in the absence of glucose. In contrast, there was little change in the level of GLUT-3 mRNA when the cells were cultured in the presence of 5 mmol/l galactose. In high concentrations of glucose, there was a decline in GLUT-3 mRNA indicating that the GLUT-3 mRNA is regulated by the extracellular concentration of glucose. In contrast, at both 5 mmol/l and 25 mmol/l glucose, the level of aldose reductase mRNA was increased. Furthermore, there were differences in the magnitude of the increase of aldose reductase mRNA between bovine retinal pericytes and bovine retinal endothelial cells with a greater increase being observed in the pericytes. We propose that this demonstration of a facilitative glucose transporter system within retinal cells, and in particular the specific response to different hexoses and the known distinct kinetic parameters of the transporter system in specific cell types, highlights the heterogeneity of hexose transport mechanisms in retinal cells. Thus, hypergalactosaemia as a model system for the study of diabetic retinopathy should be used with caution.

Changes in insulin-receptor mRNA levels in skeletal muscle and brown adipose tissue of weanling rats during fasting and refeeding.

Tissue-specific alterations in insulin sensitivity occur in response to fasting and refeeding, as part of the integrated adaptive mechanisms employed to adjust to major changes in nutritional status. In the present study the effects of fasting and refeeding on insulin-receptor, actin and myosin mRNA levels in skeletal muscle, and insulin-receptor and uncoupling-protein mRNA in brown adipose tissue of rats have been examined. Insulin-receptor mRNA levels increased markedly in both skeletal muscle and brown adipose tissue after a 40 h fast, the increase being greater in brown fat (8-fold) than in muscle (2-fold). On refeeding for 4 h, the insulin-receptor mRNA level in both tissues declined rapidly to control levels. An increase in insulin-receptor mRNA level was also observed in brown adipose tissue after a 16 h fast, although not in skeletal muscle. In contrast to the insulin-receptor mRNA, the level of the mRNA for the mitochondrial uncoupling protein declined markedly in brown adipose tissue during a 40 h fast. These results indicate that insulin-receptor mRNA levels are modulated in response to the alterations in nutritional status that occur during fasting and refeeding; this may reflect a nutritional influence on transcription of the receptor-protein gene.

Regulation of glucose transporters (GLUT-1 and GLUT-3) in human retinal endothelial cells.

The regulation of glucose transporters (GLUT-1 and GLUT-3), in terms of both mRNA and protein, in human retinal endothelial cells was investigated. The cells responded within 1 h of exposure to 5 mM glucose with an increase in the level of GLUT-3 mRNA that was due to an increase in the transcription of the 4.1 kb mRNA of the gene for GLUT-3. In the absence of glucose, the gene for GLUT-1 was not transcribed but the level of GLUT-3 mRNA was increased in these conditions and this was the result of an increase in the transcription of the 4.1 kb mRNA. The level of GLUT-1 and GLUT-3 mRNA was maximal when the cells were exposed to 15 mM glucose. These results are discussed in the light of the glucose regulatory potential of the retinal microvasculature and the implications that this may have for the mechanisms of diabetic retinopathy.

Evidence for atrial natriuretic factor induced natriuretic peptide receptor subtype switching in rat proximal tubular cells during culture.

Culture and natriuretic peptide dependent changes in the expression of the natriuretic peptides atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) and the natriuretic peptide receptors A, B, and C in primary cultures of rat proximal tubular cells were demonstrated using polymerase chain reaction analysis and cyclic guanosine monophosphate response to ANF and CNP. Freshly isolated cells expressed mRNA coding for the natriuretic peptide receptor C only, with no expression of the natriuretic peptides or the natriuretic peptide receptors A or B. At confluence natriuretic peptide receptor C expression was lost, while mRNA transcripts for both ANF and BNP and the A and B receptors became apparent. The appearance of mRNA transcripts for the natriuretic peptide receptors A and B during cell growth correspond with a significant increase in the cyclic guanosine monophosphate response to both ANF and CNP, confirming the presence of functionally active guanylate cyclase linked A and B natriuretic peptide receptors. The observed changes in peptide receptor expression during culture were preceded by changes in natriuretic peptide mRNA expression, suggesting the possibility that natriuretic peptide receptor subtype switching may be under the control of endogenous peptide release. Incubation of freshly isolated proximal tubular cells with ANF, BNP, or CNP for 3 h induced similar changes in receptor expression. Incubation with ANF induced expression of the natriuretic peptide receptor B and CNP while inhibiting natriuretic peptide receptor C. Incubation with BNP induced expression of the natriuretic peptide receptor B and CNP. Incubation with CNP induced expression of the natriuretic peptide receptors A and B and CNP. These results suggest that primary cultures of rat proximal tubular cells may experience natriuretic peptide and natriuretic peptide receptor subtype switching as they approach confluence under the control of endogenously expressed natriuretic peptides.

Cytokine regulation of RANTES production by human retinal pigment epithelial cells.

The mechanism whereby inflammatory cells gain access to the retina in posterior intraocular inflammatory disease remains unclear. The chemokine RANTES has the potential to influence the migration of memory T cells and monocytes across the blood-retinal barrier during inflammatory eye disease. We have therefore examined the production of RANTES by cultured human retinal pigment epithelial cells (RPE), which form a part of the blood-retinal barrier, in response to cytokines likely to be present in the microenvironment. IL-1 beta and TNF alpha stimulated RANTES production by these cells. IFN-gamma acted synergistically with TNF alpha to increase RANTES production. In contrast, IL-4 downregulated RANTES production stimulated by TNF alpha. RT-PCR studies showed that RANTES mRNA from RPE followed the same pattern of expression in response to cytokines as did RANTES production indicating that RANTES production was controlled at, or prior to, transcription. RANTES is produced in vitro by RPE in response to the proinflammatory cytokines IL-1 beta, TNF alpha, and IFN-gamma and is therefore likely to play a role in the development of the inflammatory eye disease endogenous posterior uveitis.

Alterations in the level of insulin receptor and GLUT-4 mRNA in skeletal muscle from rats fed a kidney bean (Phaseolus vulgaris) diet.

1. A decline in the level of circulating insulin was observed in rats fed a diet containing kidney bean. 2. Consumption of a diet containing kidney bean caused an increase in the level of mRNAs for the insulin receptor (327%) and GLUT-4 (185%) in the gastrocnemius muscle. In contrast there was only a small increase in the amount of actin mRNA (125%). Since the kidney bean-fed rats are euglycaemic the results suggest that insulin receptor and GLUT-4 mRNA levels are regulated in response to circulating insulin concentrations rather than glucose. 3. No increases in the level of insulin receptor and actin mRNA were evident in the soleus muscle of rats fed the diet containing kidney bean; however a decline was observed in the level of GLUT-4 mRNA. 4. It is proposed that a component of kidney beans, most likely the lectin phytohaemagglutinin, has systemic effects which lead to changes in expression of the insulin receptor and GLUT-4 genes and to the sensitivity of muscle to insulin.

A model system for the study of human retinal angiogenesis: activation of monocytes and endothelial cells and the association with the expression of the monocarboxylate transporter type 1 (MCT-1).

AIMS/HYPOTHESIS: The growth of retinal vessels is associated with a number of disease conditions, including diabetic retinopathy and proliferative vitreo-retinopathy. In this study we describe a model of human retinal angiogenesis and show how this may be used to explain the mechanisms that are associated with the growth of new retinal vessels. METHODS: A 4 mm diameter disc of retinal tissue was placed within a fibrin matrix and the appearance was monitored daily by light microscopy. Immunohistochemical techniques were used for the detection of, glial fibrillary acidic protein, CD68, the Ki-67 antigen, vascular endothelial growth factor, monocarboxylate transporter type 1 and von Willebrand's factor. RESULTS: Vessels were evident extending from the periphery of the explant and the activation of endothelial cells was shown by immuno-peroxidase staining of paraffin embedded sections of the explants for the expression of the Ki-67 antigen, a marker of cell proliferation. The expression of glial fibrillary acidic protein and von Willebrand's factor increased with duration in culture and the presence of activated macrophages or microglia or both was shown by positive immunoreactivity for CD68 and Ki-67 and were identified by day 3. The presence of endogenous vascular endothelial growth factor and the activation of monocarboxylate transporter type 1 by vascular endothelial growth factor, showed the involvement of specific growth factors. CONCLUSION/INTERPRETATION: The explant model provides evidence for the involvement of macrophages and glial fibrillary acidic protein activation in human retinal angiogenesis and for the expression of monocarboxylate transporter type 1, which is likely to be important in the use of lactate in the hypoxic retina.