Cd36 and molecular mechanisms of insulin resistance in the stroke-prone spontaneously hypertensive rat.

Insulin resistance is of pathogenic importance in several common human disorders including type 2 diabetes, hypertension, obesity and hyperlipidemia, but the underlying mechanisms are unknown. The spontaneously hypertensive rat (SHR) is a model of these human insulin resistance syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridemia, and hypertension map to a single region on rat chromosome 4. Genetic analysis of an SHR derived from a National Institutes of Health colony led to the identification of a causative mutation in the SHR Cd36. We have investigated glucose and fatty acid metabolism in the stroke-prone SHR (SHRSP). We demonstrate defects in insulin action on 2-deoxy-D-glucose transport (SHRSP 3.3 +/- 1.5 vs. 21.0 +/- 7.4 pmol x min(-1) x [20 microl packed cells](-1), SHRSP vs. WKY, respectively, P = 0.01) and inhibition of catecholamine-stimulated lipolysis (P < 0.05 at all concentrations of insulin) in adipocytes isolated from SHRSP. In contrast, basal levels of catecholamine-stimulated nonesterified free fatty acid (NEFA) release and plasma levels of NEFA are similar in SHRSP and WKY. These results are in agreement with the data on the SHR.4 congenic strain, which suggested that the QTL containing Cd36 mutations accounted for the entire defect in basal catecholamine action but only for approximately 40% of the SHR defect in insulin action. In the SHR, both abnormalities appear consequent of defective Cd36 expression. Because Cd36 sequence and expression are apparently normal in SHRSP, it is likely that the molecular mechanism for defective insulin action in this strain is caused by a gene(s) different than Cd36.

Increased collagen-linked fluorescence in skin of young patients with type I diabetes mellitus.

Our objective was to determine whether the fluorescence of skin collagen, which may reflect the accumulation of advanced glycosylation end products, is increased in young patients with type I (insulin-dependent) diabetes. Our study design was a cross-sectional case-control study in a referral-based diabetic clinic in an academic hospital. Study subjects comprised a convenience sample of 18 type I diabetic patients aged 17-30 yr and 8 age-matched healthy control subjects. The fluorescence of collagen was measured in skin biopsy material. Collagen-linked fluorescence (CLF) was increased in diabetic patients (mean 10.5 [range 5.8-15.8] U/mg) compared with control subjects (7.6 [5.6-10.1] U/mg, P less than 0.02). In diabetic patients, CLF was related to age (r = 0.581) and duration of diabetes (r = 0.697) but not concentration of glycosylated hemoglobin (r = 0.082). Partial correlation analysis demonstrated that duration of diabetes is the main factor determining the fluorescence of collagen in these patients. There was a relationship between CLF and presence of diabetic retinopathy after the data were adjusted for patient age and duration of diabetes (P = 0.023). Increased fluorescence of skin collagen can be detected in young type I diabetic patients and is primarily related to duration of diabetes.

Collagen-linked fluorescence in human atherosclerotic plaques.

Advanced glycosylation endproducts (AGE) are intraprotein crosslinks which form in the late stages of the Maillard (browning) reaction. It is unknown whether local changes in AGE-modified collagen occur within arteries. We measured AGE-modified collagen as collagen-linked fluorescence (CLF) in human arterial tissue and in various forms of atherosclerotic plaque. All tissues showed single fluorescence peak at excitation wavelength 340 nm and emission wavelength 420-440 nm. CLF in the aorta was 27.9 +/- 8.5 units/mg, in the coronary arteries 25.9 +/- 6.3 units/mg and in the tendon 47.8 +/- 11.5 units/mg. CLF in the skin correlated with CLF in the aorta (r = 0.467, P = 0.025) but not with CLF in coronary arteries (P = 0.935). In areas of aorta covered by superficial plaque, CLF was decreased compared with adjacent, atheroma-free segments (22.2 +/- 5.2 units/mg vs. 27.9 +/- 8.5 units/mg; P = 0.01). The CLF of collagenous plaques correlated with CLF of the atheroma-free regions. Individuals with low to moderate atheroma had lower (20.0 units/mg) CLF in superficial atherosclerotic plaques than patients with severe atheroma (22.5 units/mg; P = 0.0466). Our results indicate that local changes in vascular AGE-collagen concentration occur in atherosclerosis. This finding may have pathogenetic significance in atherosclerosis.

Chronic exposure of bovine aortic endothelial cells to native and oxidized LDL modifies phosphatidylinositol metabolism.

The aim of this study was to investigate the effects of chronic exposure to low density lipoprotein (LDL) and oxidised LDL (OXLDL) on phosphatidylinositol metabolism in bovine aortic endothelial cells. Basal levels of total inositol phosphates and inositol 1,4,5-trisphosphate were increased after both 18 and 66 h exposure to OXLDL 20 micrograms/ml. Levels also tended to be increased after exposure to LDL but this only reached significance for LDL 20 micrograms/ml after 18 h exposure. Absolute levels of inositol phosphates after stimulation with ATP were unaffected by incubation with LDL or OXLDL. However, when expressed as a percentage of basal levels, stimulated levels of inositol phosphates were reduced for ATP 10(-3) and 10(-4)M. Uptake of [3H]inositol into the phosphatidylinositol cycle was reduced after incubation with LDL and OXLDL for either 18 or 66 h. The effect of OXLDL was greater than that of LDL. The antioxidants EDTA and N-acetylcysteine attenuated the effects of LDL but not OXLDL. In addition, catalase but not mannitol or superoxide dismutase modified the effect of LDL on [3H]inositol uptake. These studies show that chronic exposure to OXLDL and to a lesser extent LDL can modify phosphatidylinositol metabolism in bovine aortic endothelial cells and that the effects of LDL may be attenuated by antioxidants and free radical scavengers. We hypothesise that the decreased uptake of [3H]inositol could be related to an alteration in membrane structure and integrity and may reflect alteration in transport of a number of ions and molecules.

Oxidized-LDL induced changes in membrane physico-chemical properties and [Ca2+]i of bovine aortic endothelial cells. Influence of vitamin E.

The effects of 3 days' exposure to native and oxidatively modified human low density lipoprotein (LDL and Ox-LDL) on cultured bovine aortic endothelial cell cholesterol content, membrane microviscosity and intracellular free calcium concentration ([Ca2+]i) were studied. Free cholesterol content increased by 35% and 100% in LDL and Ox-LDL treated cells, respectively, these effects being reversed by vitamin E; esterified cholesterol, which rose by 110% in the Ox-LDL group only, was not affected by vitamin E. Membrane microviscosity, measured as the fluorescence polarization of the trimethylammonium derivative of diphenyl-hexatriene, increased by 9% in Ox-LDL treated cells only. This effect was also reversed by vitamin E. Using the calcium sensitive fluorescent dye fura 2-AM, increases in basal [Ca2+]i of 36% in LDL and 81% in Ox-LDL treated cells were observed. The bradykinin mediated increase in [Ca2+]i was enhanced in both the LDL and, to a greater extent, the Ox-LDL group. Vitamin E reversed the effects of LDL on [Ca2+]i but had no influence in the Ox-LDL group. The lipoproteins affected all parameters measured in this study. Oxidized LDL produced reversible and irreversible alterations to the membrane and the [Ca2+]i. All changes associated with LDL were abolished by vitamin E. Such modifications in the physicochemical properties of the membrane and [Ca2+]i could be involved in the initiation of the atherosclerotic process.

The effects of ageing on glycation and the interpretation of glycaemic control in Type 2 diabetes.

To investigate the discrepancy in the assessment of glycaemic control using glycated haemoglobin (HbA1C) and glycated proteins (fructosamine), the effect of age on these variables was measured in non-diabetic individuals. In 232 non-diabetics, there was a linear relationship between HbA1C and age (r = 0.49, p < 0.0001). Mean HbA1C rose from 3.82% to 4.44% between the ages of 20 and 70. Consequently, when Type 2 diabetic patient samples (n = 128, median age 63 years) were classified according to European guidelines into good or poor glycaemic control using both an age-matched (n = 101) and a younger (n = 108, median age 37 years) non-diabetic reference population, fewer patients were in good control (14% vs. 25%) and more in poor control (73% vs. 53%) when the younger reference population was used (both p < 0.05). In a subgroup of 126 non-diabetic subjects, HbA1C rose with age (r = 0.48), but serum fructosamine and fasting glucose did not (r = 0.07, r = 0.009, respectively, p = NS). Age-associated differences in non-diabetic HbA1C values may affect the assessment of glycaemic control in diabetic patients. It may also partly explain discrepancies found when comparing fructosamine with HbA1C as a measure of glucose control. Age-related HbA1C reference intervals may therefore be required for the treatment of patients and the accurate auditing of clinic performance.

The effect of hypoalbuminaemia, hyperbilirubinaemia and renal failure on serum fructosamine concentration in non-diabetic individuals.

We have investigated the effects of hypoalbuminaemia, hyperbilirubinaemia and renal failure on serum fructosamine concentration in 39 non-diabetic patients. All patients were hypoalbuminaemic (median serum albumin 25 g/l, range 12-34 g/l). Group 1 (n = 19) were patients with hypoalbuminaemia alone, group 2 (n = 7) with hypoalbuminaemia and impaired renal function (median serum creatinine 226 mumol/l, range 154-461 mumol/l) and group 3 (n = 13) were subjects with hypoalbuminaemia and hyperbilirubinaemia (median serum bilirubin 34 mumol/l, range 19-83 mumol/l). Serum fructosamine was significantly lower in all three groups compared to age-matched normoalbuminaemic controls, but there was no significant difference in fructosamine concentrations between the groups. There was a correlation between fructosamine concentration and serum albumin. (r = 0.82, p less than 0.001) in all three groups combined. Serum fructosamine did correlate with serum bilirubin in patients with normal renal function (r = 0.0, p less than 0.001). In patients with abnormal renal function there was no correlation between serum fructosamine and either urea (r = 0.22, ns) or creatinine (r = 0.31, ns). Albumin is the major factor affecting serum fructosamine concentrations. Moderate hyperbilirubinaemia does not affect fructosamine concentration. No difference in fructosamine concentration could be demonstrated in patients with renal failure.

The effect of delta-gluconolactone, an oxidised analogue of glucose, on the nonenzymatic glycation of human and rat haemoglobin.

Nonenzymatic glycation of proteins and oxidative stress are considered independent factors important in the development of the complications of diabetes but may be interrelated by the process of autoxidative glycation. This pathway involves monosaccharide autoxidation to a reactive ketoaldehyde analogue and subsequent reaction with protein to form a ketoimine adduct. This study demonstrates that delta-gluconolactone (delta-GL), an oxidised analogue of glucose, is a potent glycating agent in vitro of haemoglobin present in blood samples from insulin-dependent diabetic and non-diabetic human subjects and from spontaneously diabetic, insulin-dependent BB/Edinburgh (BB/E) rats. The percentage glycated haemoglobin after incubation (37 degrees C, 5 h) with delta-GL (25 mmol/l) was significantly (P < 0.002) higher than that observed using an equimolar concentration of glucose. Intravenous administration of delta-GL (1 g/kg) to non-diabetic BB/E rats also significantly increased glycation of haemoglobin (6.0 +/- 0.1% vs 4.9 +/- 0.1%, P < 0.01) whereas intravenous injection of an identical dose of glucose had no significant effect (5.1 +/- 0.1% vs 5.0 +/- 0.2%). These results support the hypothesis that nonenzymatic glycation of proteins involves attachment by both native and oxidised monosaccharides. Further investigation of the interactions between diabetes-associated increases in oxidative stress and glycation on the development and progression of the vascular complications of diabetes is necessary.

Serum fructosamine/haemoglobin A1 ratio predicts the future changes in haemoglobin A1 in type 2 (non-insulin dependent) diabetic patients.

The aim of the study was to define clinical interpretation of the parallel measurements of serum fructosamine and HbA1 in diabetic patients. We studied 14 type 2 diabetic patients over a 16-wk period. The cross-sectional analysis showed no correlation between serum fructosamine and HbA1 concentrations during the period of changing glycaemic control. The correlations, however, became significant (P less than 0.05) at 12 (r = 0.60) and 16 (r = 0.87) weeks, i.e. after glycaemia had stabilised. Longitudinal analysis of individual patients' data over the 16-wk period showed a significant correlation between serum fructosamine and HbA1 (r = 0.55 to r = 0.94) which was present in 8 out of 14 patients. The changes in fructosamine concentration preceded those observed in HbA1. The ratio of fructosamine/HbA1 predicted the changes in HbA1 over the following month (r = 0.54, P less than 0.001). Thus, we demonstrated that the parallel measurement of fructosamine and HbA1 provides information on future trends in HbA1 concentration in diabetic patients.

The measurement of lipoprotein subfractions in plasma using a tabletop ultracentrifuge.

We adapted the ultracentrifugation method of the Lipid Research Clinics Program for the separation of lipid subfractions (LDL, VLDL and HDL cholesterol) to a tabletop ultracentrifuge (Beckman TL-100). Centrifugation time was reduced from 18 h to 2.5 h and the sample volume from 5 mL to 2 mL plasma. The imprecision of the LDL-cholesterol estimation (coefficient of variation = CV) was 2.9-7.4% and that of HDL-cholesterol measurement was 1.4-3.9%. Imprecision of the VLDL-C measurement was high (CV = 15.6-29.8%). The results correlated with those obtained by the Lipid Research Clinics method (P less than 0.001). Our method could be conveniently adapted by clinical laboratories serving specialist lipid clinics.

Screening for hyperlipidaemia in diabetes mellitus. Relationship to glycaemic control.

Diabetic patients have an increased risk of developing cardiovascular disease which, in part, may be due to lipid abnormalities. Our aim was to establish from an initial screening programme what proportion of diabetic patients attending a routine diabetic outpatient clinic had hyperlipidaemia despite having good or acceptable glycaemic control. We screened 299 randomly selected diabetic patients to assess the prevalence of hyperlipidaemia and its relationship to glycaemic control. Twenty-eight per cent had hyperlipidaemia (defined as cholesterol greater than 6.5 mmol/L and/or non-fasting triglycerides greater than 3 mmol/L). Of these hyperlipidaemic patients, 71% had good or acceptable glycaemic control as defined by a glycated haemoglobin value of less than 10%. Approximately 40% of type 2 diabetic patients had body mass index values outside recommended targets indicating the potential of weight reduction in this group as a treatment modality. Our results indicate that the majority of hyperlipidaemic diabetic patients had good or acceptable glycaemic control, and as such these patients are potential candidates for specific lipid lowering therapy.

Long-term performance of the fructosamine assay.

We have investigated the long-term performance of the fructosamine assay based on secondary glycated protein standards and attempted to define the interpretation of varying degrees of increase in fructosamine concentration in comparison to haemoglobin A1 (HbA1) values both in insulin dependent (IDDM) and non-insulin dependent (NIDDM) diabetic patients. Between-batch imprecision of fructosamine over 5 months was (CV) 2.5% at 2.09 mmol/L, 2.8% at 3.52 mmol/L and 3.6% at 4.14 mmol/L. Variation of fructosamine concentration in vivo in stable diabetic patients monitored over 8-18 weeks was 2.3% to 7.1%. Fructosamine correlated with HbA1 both in IDDM (n = 110, r = 0.701, P less than 0.001) and NIDDM (n = 71, r = 0.764, P less than 0.001). Specificity and sensitivity of fructosamine for the prediction of degree of control assessed on the basis of HbA1 level (cut-off point for good vs. poor control, HbA1 = 10%) was determined. In NIDDM, specificity above 90% was achieved at a fructosamine concentration of 3.4 mmol/L with a corresponding sensitivity of 64.1%. 22.5% of patients were classified differently on the basis of fructosamine as compared to HbA1. In IDDM, specificity over 90% was achieved at 3.8% mmol/L fructosamine with a sensitivity of 35%. Discordancy rate between HbA1 and fructosamine based assessment of control was 31.8%. The assessment of diabetic control based on fructosamine may be different from that based on HbA1, particularly in IDDM. Fructosamine and HbA1 should be used as complementary rather than alternative tests.

Low-density lipoprotein particle size in type 2 diabetic patients and age matched controls.

Non-enzymatic glycation of low-density lipoprotein (LDL), and the predominance of small dense LDL particles may together contribute to the increased risk of atherosclerosis in diabetes. We aimed to establish whether the size of LDL particles is related to plasma triglyceride concentration, and to the extent of LDL glycation in type 2 diabetic patients. Sixteen men with type 2 diabetes and 16 age matched non-diabetic controls were studied. LDL size was measured by rapid density gradient ultracentrifugation, and LDL glycation by affinity chromatography. Modal LDL density correlated with plasma triglyceride concentrations in both diabetic and control groups (r = 0.86, P < 0.0001, and r = 0.76, P < 0.0008, respectively). There was no significant difference in these variables between the groups. LDL modal density showed no correlation with HbA1, serum fructosamine or plasma glucose in either group. In the diabetic group the degree of LDL glycation correlated with serum fructosamine (r = 0.74, P < 0.001), HbA1 (r = 0.65, P < 0.008), and with plasma glucose (r = 0.64, P < 0.008). Our results suggest that, in well controlled type 2 diabetic patients LDL size is independent of short-term glycaemic control but can be predicted by plasma triglyceride concentrations.

Bone density and antiepileptic drugs: a case-controlled study.

This case-controlled study explored the relationship between bone mineral density (BMD) and long-term treatment with antiepileptic drugs (AEDs) in older adults with epilepsy. Seventy-eight patients (47 post-menopausal females, 31 males, aged 47-76 years) with epilepsy participated in the study. Each had only ever received treatment with either enzyme-inducing (n = 52) or non-inducing (n = 26) AEDs. Individuals were matched for age, sex, height and weight with a drug-naive control. All patients underwent bone densitometry at the lumbar spine and femoral neck and had blood sampling and urine collected for a range of bone markers. Male patients had lower BMD than controls at the lumbar spine (P < 0.01) and neck of the femur (P < 0.005). Female patients had significantly reduced bone density at the femoral neck (P < 0.05) only. AED usage was independently associated with an overall reduction in bone density at femoral sites and contributed to just over 5% of the variance at the femoral neck. Duration of treatment and type of AED were not independent factors for reduction in BMD. This case-controlled study supports the hypothesis that long-term AED therapy is an independent risk factor for reduced BMD in epileptic patients. Adults receiving treatment for epilepsy are at higher risk of osteoporosis and should be offered bone densitometry.

Glycated haemoglobin values: problems in assessing blood glucose control in diabetes mellitus.

OBJECTIVE: To see whether two measures of glycated haemoglobin concentration--the haemoglobin A1 (HbA1) value and the haemoglobin A1c (HbA1c) value--assess blood glucose control differently in diabetes. DESIGN: Diabetic patients had glycaemic control assessed on the basis of HbA1 and HbA1c values measured by the same high performance liquid chromatography instrument and on the basis of HbA1 measured by electrophoresis. SETTING: A diabetic outpatient clinic. SUBJECTS: 208 diabetic patients and 106 non-diabetic controls. MAIN OUTCOME MEASURES: Glycated haemoglobin concentrations classified according to European guidelines as representing good, borderline, or poor glycaemic control by using standard deviations from a reference mean. RESULTS: Fewer patients were in good control (25;12%) and more poorly controlled (157;75%) as assessed by the HbA1c value compared with both HbA1 assays (39 (19%) and 130 (63%) respectively when using high performance liquid chromatography; 63 (30%) and 74 (36%) when using electrophoresis). The median patient value was 8.0 SD from the reference mean when using HbA1c, 5.9 when using HbA1 measured by the same high performance liquid chromatography method, and 4.1 when using HbA1 measured by electrophoresis. CONCLUSIONS: Large differences exist between HbA1 and HbA1c in the classification of glycaemic control in diabetic patients. The HbA1c value may suggest a patient is at a high risk of long term diabetic complications when the HbA1 value may not. Better standardisation of glycated haemoglobin measurements is advisable.

Risk factors for coronary disease: the time for a paradigm shift?

Risk stratification is a key element of clinical management not only in the primary and secondary prevention, but also during the acute stages of cardiovascular disease. The current risk assessment algorithms in primary prevention are based on established risk factors: gender and age, cigarette smoking, the presence of hypertension and diabetes mellitus, and serum concentrations of total cholesterol, low-density lipoprotein (LDL)-cholesterol and high-density lipoprotein-cholesterol. However, many individuals who are assessed as "low risk" on the basis of traditional risk factors, still develop cardiac events. This article addresses current issues relevant to the assessment of cardiovascular risk. It emphasizes the potential importance of disturbed energy supply for atherogenesis, by introducing the concept of fuel transport (chylomicron, VLDL, and remnants) and overflow (LDL) pathways of lipid metabolism. It highlights the present lack of routine methods to monitor the fuel transport pathway. It considers the measurements of serum C-reactive protein and plasma fibrinogen as new additions to the cardiovascular risk factor profiles. Finally, risk stratification based on the traditional and the new risk factors is linked to that based on the markers of acute myocardial damage such as cardiac troponin I or troponin T. It is concluded that the combined use of the markers of myocardial damage and the "new" cardiovascular risk factors is the way ahead for the assessment of cardiovascular risk.