Rachel M. Knott

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.

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

SummaryThe 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 facultative 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.

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.

A potential new prodrug for the treatment of cystinosis: Design, synthesis and in-vitro evaluation

Nephropathic cystinosis is a rare autosomal recessive disease characterised by raised lysosomal levels of cystine in the cells of most organs. The disorder is treated by regular administration of the aminothiol, cysteamine, an odiferous and unpleasant tasting compound that along with its metabolites is excreted in breath and sweat, leading to poor patient compliance. In an attempt to improve patient compliance a series of novel prodrugs has been designed and evaluated as a potential new treatment for nephropathic cystinosis. The first of the prodrugs tested, 3a, was found to decrease the levels of intracellular cystine in cystinotic fibroblasts. This is the first report of a potential new therapeutic treatment for nephropathic cystinosis since the advent of cysteamine bitartrate.

Regulation of plasminogen activation by TGF-β 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 β (TGF-β) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-β 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-β (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-β resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-β 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-β increases PAI-1 and decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.

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.

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.

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 respone 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.

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.

The hen's egg chorioallantoic membrane (HET-CAM) test to predict the ophthalmic irritation potential of a cysteamine-containing gel: Quantification using Photoshop® and ImageJ.

A modified hens egg chorioallantoic membrane (HET-CAM) test has been developed, combining ImageJ analysis with Adobe(®) Photoshop(®). The irritation potential of an ophthalmic medicine can be quantified using this method, by monitoring damage to blood vessels. The evaluation of cysteamine containing hyaluronate gel is reported. The results demonstrated that the novel gel formulation is non-irritant to the ocular tissues, in line with saline solution (negative control). In conclusion, the modification of the established HET-CAM test can quantify the damage to minute blood vessels. These results offer the possibility to formulate cysteamine in an ocular applicable gel formulation.