Elizabeth Trimble

The effect of Endothelin 1 on the retinal microvascular pericyte

The effect of the highly vasoactive peptide endothelin 1 (ET1) was tested on bovine retinal microvascular pericytes propagated in vitro. Specific binding of 125I-ET1 to retinal pericytes was documented by autoradiography. ET1 caused contraction of pericytes at a concentration of 0.1 nM which was accompanied by increases in inositol phosphates. Exposure of pericytes to 10 nM ET1 resulted in the aggregation and realignment of muscle-specific actins into bundles which were oriented parallel to the long axis of the cell, and ET1 was also mitogenic to pericytes in the presence of low levels of fetal calf serum. These observations suggest that ET1 may play an important role in endothelial cell-pericyte interactions within the microvasculature of the retina and that it may be involved in the autoregulation of retinal blood flow.

Very low density lipoprotein subfractions in Type II diabetes mellitus: alterations in composition and susceptibility to oxidation

Aims/hypothesis. Type II (non-insulin-dependent) diabetes mellitus is associated with raised triglycerides and increased very low density lipoprotein cholesterol. The aim of this study was to assess if very low density lipoprotein subfraction composition and potential to oxidise were altered in this condition.¶Methods. Very low density lipoprotein was separated into four subfractions (A→D) by a novel, rapid ultracentrifugation procedure. Analysis of each subfraction included lipid and fatty acid composition. Preformed peroxides were measured spectrophotometrically and conjugated dienes were used as an indicator of in vitro lipid oxidation.¶Results. In all results we compared patient and control subfractions. Mean fasting plasma glucose was 8.9 ± 2.0 mmol/l in patients vs 5.1 ± 0.4 mmol/l in control subjects (p < 0.001); patient HbA1 c was 7.6 ± 1.4 %. Patient total lipid standardised for apo B was higher than controls in subfractions A, B and C; A, 201 vs 60; B, 191 vs 40; C, 63 vs 21; D, 29 vs 34 μmol lipid per mg apo B (p < 0.05). Preformed peroxides were higher in all patient subfractions compared with controls: A, 340 vs 48; B, 346 vs 42; C, 262 vs 28; D, 54 vs 16 nmol per mg apo B (p < 0.001). Patient subfractions A and D were more susceptible to in vitro oxidation. Monounsaturated fatty acids were lower in patients subfractions, 35.2 vs 36.7; B, 35.1 vs 38.7; C, 34.4 vs 36.5; D, 33.0 vs 35.5 as per cent total (p < 0.05).¶Conclusions/interpretation. These results indicate abnormalities in very low density lipoprotein subfraction composition and oxidation profile in Type II diabetic subjects, which are characteristic of more atherogenic particles and that may contribute to the development of cardiovascular disease in these patients. [Diabetologia (2000) 43: 485–493]

Underestimation of glucose turnover determined using [6-3H]glucose tracer in non-steady state

SummaryThe use of tritiated glucose tracers may result in underestimation of glucose turnover during hyperinsulinaemic clamps giving paradoxical negative endogenous glucose production rates. While mathematical modelling errors in the analysis of tracer data are major determinants of this underestimate in the non-steady state, the relative importance of tracer contamination under these conditions remains in doubt. We have used high performance liquid chromatography to assess the possible contribution to this problem of a labelled tracer impurity found in [6-3H]glucose. In conventional 4 h hyperinsulinaemic clamps performed in six normal subjects, labelled impurity increased as a percentage of the neutral plasma radioactivity fraction from 5.3±0.9% after a 2 h equilibration period (0 min) to 13.5±2.2% at 120 min and 15.4±2.4% at 240 min, as plasma glucose specific activities fell following the infusion of insulin. Negative endogenous glucose production rates were observed both at 90–120 min (−8.8±1.6μmol·kg−1min−1) and at 210–240 min (−8.5±1.4 μmol·kg−1min−1) implying a persistent underestimate in isotopically determined glucose appearance rate. Using chromatography data to correct for impurity increased glucose appearance rates by 7.9±2.1% at 120 min and 11.0±2.5% at 240 min. Purified tracer was then used for a further six clamps. When the conventional protocol was used with unlabelled glucose infusion an obvious negative error persisted only at 90–120 min. In contrast, labelled infusions gave exclusively positive values for endogenous glucose production. We conclude that a labelled impurity of [6-3H]glucose may be an important source of error in measurement of glucose turnover and endogenous glucose production in the non-steady state. Use of chromatographically pure tritiated glucose tracers is recommended.

Altered endothelin-1 induced contraction and second messenger generation in bovine retinal microvascular pericytes cultured in high glucose medium.

SummaryThe effect of simulated hyperglycaemia on bovine retinal pericytes was studied following culture of these cells for 10 days under normal (5 mmol/l) and elevated (25 mmol/l) glucose conditions in the absence of endothelial cells. Pericytes cultured under high ambient glucose exhibited both a delayed and reduced contractile response following stimulation with endothelin-1. Stimulation with 10−7mol/l endothelin-1 for 30 s caused significant contraction in cells grown in both 5 mmol/l and 25 mmol/l glucose. The former also contracted significantly with 10−8mol/l endothelin-1. Further, at all concentrations tested, statistical comparison of the time course of contraction showed a significant difference (p<0.02) in the reduction of planimetric surface area between the two cell groups. Since neither binding of endothelin-1 nor the number of receptors for this peptide were significantly different (p>0.1) between bovine retinal pericytes grown for 10 days under normo- or hyperglycaemic conditions, it became apparent that the altered contractility in bovine retinal pericytes following culture in high glucose must be due to post-binding intracellular disturbance(s). Indeed, both basal and 15 s post-stimulation with 10−8 mol/l endothelin-1, levels of inositol trisphosphate were significantly reduced (p<0.05 and p<0.02, respectively) in pericytes cultured for 10 days in 25 mmol/l glucose. These results show that endothelialindependent alterations in contractility of pericytes occur when they are grown in conditions which simulate hyperglycaemia. The results also suggest that the observed attenuation in response to endothelin-1 stimulation evident in pericytes grown under simulated hyperglycaemic conditions is not due to alterations in peptide binding.

Contribution of glucose/glucose 6-phosphate cycle activity to insulin resistance in Type 2 (non-insulin-dependent) diabetes mellitus

SummaryIt has been suggested that increased glucose/glucose 6-phosphate substrate cycling impairs net hepatic glucose uptake in Type 2 (non-insulin-dependent) diabetes mellitus and contributes to hyperglycaemia. To investigate glucose/glucose 6-phosphate cycle activity and insulin action in Type 2 diabetes we studied eight patients and eight healthy control subjects, using the euglycaemic glucose clamp and isotope dilution techniques with purified [2-3H]- and [6-3H] glucose tracers, in the post-absorptive state and eight patients and five healthy control subjects during consecutive insulin infusions at rates of 0.4 and 2.0 mU·kg−1·min−1. [2-3H]glucose and [6-3H]glucose radioactivity in plasma samples were determined using selective enzymatic detritiation, allowing calculation of glucose turnover rates for each isotope, the difference being glucose/glucose 6-phosphate cycling. Endogenous glucose production ([6-3H]glucose) was greater in diabetic than control subjects in the post-absorptive state (15.6±1.5 vs 11.3±0.4 μmol·kg−1·min−1, p<0.05) and during the 0.4 mU insulin infusion (10.1±1.3 vs 5.2±0.3 μmol·kg−1·min−1, p<0.01) indicating hepatic insulin resistance. Glucose/glucose 6-phosphate cycling was significantly greater in diabetic than in control subjects in the post-absorptive state (2.6±0.4 vs 1.6±0.2 μmol·kg−1·min−1, p<0.05) but not during the 0.4 mU insulin infusion (2.0±0.4 vs 2.0±0.3 μmol·kg−1·min−1). During the 2.0 mU insulin infusion endogenous glucose production was suppressed to a similar degree in both groups (2.6±0.5 vs 3.4±0.7 μmol · kg−1·min−1) but glucose disappearance was lower in the diabetic subjects (30.8±2.0 vs 52.4±4.6 μmol·kg−1·min−1, p<0.01). During the 2.0 mU insulin infusion glucose/glucose 6-phosphate cycling was greater in the diabetic subjects (3.8±0.7 vs 0.8±0.6 μmol·kg−1·min−1, p<0.05). In conclusion, both hepatic and peripheral insulin action are impaired in Type 2 diabetes. Increased glucose/glucose 6-phosphate cycling is seen in the post-absorptive state and also during marked hyperinsulinaemia, when insulin resistance is predominantly due to reduced peripheral tissue glucose uptake.