Crush injury to motor nerves in the G93A transgenic mouse model of amyotrophic lateral sclerosis promotes muscle reinnervation and survival of functionally intact nerve-muscle contacts.

Selective survival of small motor nerve fibers and their neuromuscular contacts in the SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS) suggests that smaller regenerated nerve fibers are more able to sustain reformed nerve-muscle connections as functionally intact motor units (MUs). The sciatic nerve was crushed unilaterally in SOD1G93A transgenic mice at 40 days of age and contractile forces of reinnervated muscles and their MUs were recorded at 90 days in order to determine the capacities of the nerves to regenerate and to form and retain functional neuromuscular connections. Reduced MU numbers in fast-twitch tibialis anterior, extensor digitorum longus and medial gastrocnemius muscles and the lesser reductions in slow-twitch soleus muscle of SOD1G93A transgenic mice were reversed in reinnervated muscles: there were more reinnervated MUs and their contractile forces and the muscle forces and weights increased. In line with the contrasting ability of only small not large nerve fibers to sprout to form enlarged MUs in the SOD1G93A transgenic mouse, the smaller regenerating nerve fibers formed enlarged MUs that were better able to survive. Because nerve fibers with and without muscle contacts were severed by the sciatic nerve crush injury, the conditioning lesion is untenable as the explanation for improved maintenance of reinnervated neuromuscular junctions. Elevated neurotrophic factor expression in axotomized motoneurons and/or denervated Schwann cells and the synapse withdrawal from axotomized motoneurons are other factors that, in addition to reduced size of nerve fibers reinnervating muscles, may account for increased survival and size of reinnervated MUs in ALS.

Ethnic differences in retinal microvascular structure.

AIMS/HYPOTHESIS: People of African origin have increased risk of stroke and retinal microvascular disease compared with populations of European origin. We compared quantitative measures of retinal microvasculature in British white Europeans and African Caribbeans. METHODS: Population-based study of 215 (45% male) British African-Caribbean migrants and 323 (48% male) white Europeans aged 40-69 years. Digitised retinal images were analysed using a validated semi-automated system. RESULTS: Arteriolar optimality deviation, an indicator of endothelial dysfunction, was greater in African Caribbeans (age- and sex-adjusted means [95% CIs]: 0.06 [0.05-0.06] vs 0.04 [0.04-0.05], p = 0.004); this was unexplained by conventional risk factors. Arteriolar diameters were narrower in African Caribbeans (age- and sex-adjusted means [95% CIs]: 18.4 [18.1-18.6] vs 17.9 [17.6-18.2], p = 0.011). These ethnic differences in diameters were attenuated on adjustment for systolic BP (SBP) (adjusted means: 18.2 vs 18.1, p = 0.31). However, there was a significant interaction (p = 0.011) between diabetes and SBP, such that SBP was strongly associated with arteriolar diameter in people without diabetes, but not in those with diabetes (adjusted beta-coefficients for SBP: Europeans: -0.42, p = 0.002 vs 0.17, p = 0.69, African Caribbeans: -0.35, p = 0.023 vs 0.01, p = 0.96). Other measures of retinal vasculature did not differ by ethnicity. CONCLUSIONS/INTERPRETATION: British African Caribbeans appear to have poorer retinal arteriolar endothelial function than white Europeans. Higher BPs explained the narrower arterioles in African Caribbeans; however, patterns of association between arteriolar narrowing and BP suggest the possibility that cerebral autoregulation and/or remodelling might be adversely affected by diabetes in both ethnic groups.

The effect of peripheral nerve injury on disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Around 20% of familial cases of amyotrophic lateral sclerosis have been shown to carry mutations in Cu/Zn superoxide dismutase 1 (Cu/Zn SOD1). Transgenic mice over-expressing human mutant SOD1 genes have been developed and in this study we examined the effect of nerve injury on disease progression in these mice. Firstly, disease progression in uninjured mice was characterised using physiological methods. Muscle force, contractile characteristics and motor unit survival was established at 90 days, an early symptomatic stage and also at the end-stage of the disease, at 130 days. In addition, muscle histochemistry was examined and the extent of motoneuron survival established morphologically. By 90 days of age, there is a significant reduction in muscle force, and nearly 40% of motoneurons within the sciatic motor pool have already died. By 130 days, the muscles are significantly weaker, and there is a dramatic change in the phenotype of extensor digitorum longus (EDL), which changes from a fast fatigable muscle, to a fatigue resistant muscle with a high oxidative capacity. By this stage of the disease, only 40% of motor units in EDL survive, with only 29% of motoneurons surviving within the sciatic motor pool. Following injury to the sciatic nerve in SOD1(G93A) mice, there is an acceleration in disease progression so that 90 day old mice show deficits that are only seen at the end stage in uninjured SOD1(G93A) mice. It is therefore possible that mutant SOD1 toxicity increases the vulnerability of motoneurons and muscles to stressful stimuli such as nerve injury.

Serum levels of low molecular weight advanced glycation end products in diabetic subjects.

AIMS: One of the principal theories of the development of diabetic complications proposes that increased levels of advanced glycation end products (AGE) are formed in diabetes by prolonged exposure of proteins, lipids and nucleotides to glucose. Such AGEs may contribute to the development of diabetic complications by a number of mechanisms. Circulating AGEs can be detected in serum, and in the present study, we analysed the clinical correlates of circulating serum low molecular weight AGE (LMW-AGE). METHODS: Serum LMW-AGE was measured in 106 non-diabetic and 499 diabetic subjects using fluorescence spectroscopy. Results were calibrated against an in-house AGE albumin preparation, and expressed as absolute fluorescence units (AFU). RESULTS: Serum LMW-AGE values were significantly higher in diabetic than non-diabetic subjects [median 7.5 (range 0-595.5) vs. 5.3 (1.0-15.5) AFU, P<0.01]. In the normal subjects, there were significant correlations between serum LMW-AGE and age (r=0.42, P<0.01) and serum creatinine (r=0.39, P<0.01). In the diabetic patients, serum LMW-AGE correlated significantly with age (r=0.315, P<0.01), systolic blood pressure (r=0.141, P=0.002), serum creatinine (r=0.449, P<0.01) and urinary albumin/creatinine ratio (ACR) (r=0.265, P<0.01). There was no correlation between serum LMW-AGE and HbA1c. On regression analysis, with serum LMW-AGE as the dependent variable, serum creatinine emerged as the most significant factor (t=8.1, P<0.01), followed by age (t=4.0, P<0.01) and ACR (t=2.9, P=0.004). There was no significant difference in serum LMW-AGE between those with and without retinopathy or in those with vascular disease. CONCLUSIONS: We conclude that circulating LMW-AGEs are increased in diabetic subjects. The major determinant appears to be renal dysfunction in the form of raised albumin/creatinine ratio or creatinine. There was no association with other markers of vascular disease or presence of diabetic complications.

The role of growth factors in the development of diabetic retinopathy.

Proliferation of retinal blood vessels is one of the most striking features of advanced diabetic retinopathy. This feature has led to the conclusion that the normal balance of growth factors, which usually serves to keep angiogenesis in check, is disturbed in diabetic retinopathy. A considerable amount of work has been performed in the field of angiogenesis within the last decade. Much of this is applicable to diabetic eye disease, but due to the lack of an animal model, few studies have been performed directly on models of diabetic retinopathy. This review examines the literature as it relates to diabetic retinopathy.

Hypertensive retinopathy in Afro-Caribbeans and Europeans. Prevalence and risk factor relationships.

The prevalence of hypertension is particularly high in people of black African descent throughout the world, and the consequences of hypertension, such as hypertensive heart and renal disease and stroke, are also more common. But there is little consensus on whether hypertensive retinopathy follows a similar pattern. We determined the prevalence of hypertensive retinopathy and its relationships with resting and ambulatory blood pressure in a population study of Afro-Caribbeans and Europeans aged 40 to 64 years in London, UK. Retinal photographs of 651 participants were graded for hypertensive retinopathy. Age- and sex-standardized prevalence of retinopathy was 11% (95% confidence interval, 8% to 14%) in Europeans and 21% (95% confidence interval, 16% to 26%) in Afro-Caribbeans (P < .001), respectively. This ethnic difference in prevalence was greatest in normotensive women (8% in Europeans versus 20% in Afro-Caribbeans, P < .001). Resting systolic pressure was 8 mm Hg higher in normotensive Afro-Carribean compared with European women, but this could not fully account for the ethnic difference in the prevalence of retinopathy. Examination of the different relationships of age and resting and ambulatory blood pressures with hypertensive retinopathy showed that these relationships were strongest in European women and weakest in Afro-Caribbean women. We conclude that hypertensive retinopathy is more common in Afro-Caribbeans, particularly women, and that ethnic differences in resting blood pressure cannot fully account for this. The relatively weak relationship between resting and ambulatory blood pressures and retinopathy in Afro-Caribbeans suggests that factors other than blood pressure determine the high rates of hypertensive retinopathy in this group.

Hypertensive retinopathy in Afro-Caribbeans and Europeans: prevalence and risk factor relationships

The prevalence of hypertension is particularly high in people of black African descent throughout the world, and the consequences of hypertension, such as hypertensive heart and renal disease and stroke, are also more common. But there is little consensus on whether hypertensive retinopathy follows a similar pattern. We determined the prevalence of hypertensive retinopathy and its relationships with resting and ambulatory blood pressure in a population study of Afro-Caribbeans and Europeans aged 40 to 64 years in London, UK. Retinal photographs of 651 participants were graded for hypertensive retinopathy. Age- and sex-standardized prevalence of retinopathy was 11 percent (95 percent confidence interval, 8 percent to 14 percent) in Europeans and 21 percent (95 percent confidence interval, 16 percent to 26 percent) in Afro-Caribbeans (P<.0010, respectively. This ethnic difference in prevalence was greatest in normotensive women (8 percent in Europeans versus 20 percent in Afro-Caribbeans, P<.001). Resting systolic pressure was 8 mm Hg higher in normotensive Afro-Caribbean compared with European women, but this could not fully account for the ethnic difference in the prevalence of retinopathy. Examination of the different relationships of age and resting and ambulatory blood pressures with hypertensive retinopathy showed that these relationships were strongest in European women and weakest in Afro-Caribbean women. We concluded that hypertensive retinopathy is more common in Afro-Caribbeans, particularly women, and that ethnic differences in resting blood pressure cannot fully account for this. The relatively weak relationship between resting and ambulatory blood pressures and retinopathy in Afro-Caribbeans suggests of hypertensive retinopathy in this group (AU)

Whole-body leucine turnover in adults on conventional treatment for hypopituitarism.

This study has investigated protein metabolism in adults with hypopituitarism before and after growth hormone (GH) replacement and in matched controls. Whole-body leucine turnover was measured in 16 GH-deficient adult hypopituitary patients (nine females and seven males) on standard thyroid, adrenal and sex hormone replacement and in 20 normal controls using primed continuous infusion of L-[1-13C]leucine. In seven of the patients, leucine turnover was restudied following 6 months' treatment with biosynthetic human GH (0.025-0.05 IU/kg body wt daily, with the final dose determined by patient tolerance). Compared with normal controls, hypopituitary patients had significantly reduced leucine flux (mean +/- SD: 97.8 +/- 24.9 vs 131.0 +/- 23.0 mumol.h-1 x kg-1; p < 0.001), reduced leucine incorporation into protein (80.4 +/- 20.9 vs 108.8 +/- 19.6 mumol.h-1 x kg-1; p < 0.001) and reduced leucine oxidation (17.4 +/- 4.8 vs 22.2 +/- 8.1 mumol.h-1 x kg-1; p < 0.05). Leucine turnover was similar in male and female patients. In the patients, leucine flux correlated positively with body weight (rho = 0.51, p < 0.05) and leucine incorporation in protein correlated positively with lean body mass (rho = 0.55, p < 0.05) and in male patients leucine flux correlated positively with serum insulin-like growth factor I (IGF-I) levels (rho = 0.71, p < 0.05). No significant relationship was observed with age or duration of hypopituitarism. Growth hormone replacement therapy did not produce a uniform effect on leucine metabolism. Mean values of leucine flux, oxidation and incorporation into protein increased, although the differences were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic changes and vascular risk factors in hypopituitarism.

Adults with hypopituitarism die prematurely, and the excess mortality is from vascular disease. On echocardiography we have demonstrated abnormalities of myocardial diastolic function in hypopituitary adults, indicating possible early ischaemic change. Peripheral arterial disease is evident on ultrasonography. Vascular risk factors have also been examined. Impaired glucose tolerance and unrecognized diabetes are common in hypopituitary adults. Total cholesterol levels are elevated, particularly in hypopituitary women. The role of growth hormone (GH) deficiency in the vascular disease and in the vascular-risk-factor abnormalities is unknown at present. Prolonged GH therapy causes a decrease in the levels of fasting total cholesterol, without any adverse effects on glucose homeostasis. GH therapy trials in adults will clarify the role of GH in the excess vascular risk of hypopituitarism. Prolonged GH therapy will be necessary for the vascular effects to be defined.

Myocardial dysfunction in treated adult hypopituitarism: a possible explanation for increased cardiovascular mortality.

OBJECTIVE: To assess cardiac structure and function in patients with treated hypopituitarism and to determine their relation to the degree of growth hormone deficiency and body composition pattern. DESIGN: 26 patients with treated hypopituitarism were studied by cross sectional and Doppler echocardiography and by exercise testing. The results were analysed and their relation to the degree of growth hormone deficiency and body composition determined. SETTING: All tests were performed in the department of cardiology and the unit of metabolic medicine at a tertiary referral centre. PATIENTS: Patients with hypopituitarism referred for endocrine assessment. MAIN OUTCOME MEASURES: Left ventricular mass, left ventricular diastolic function, and exercise capacity in patients with hypopituitarism and their relation to growth hormone deficiency. RESULTS: Mean (SD) serum concentration of insulin-like growth factor 1 (IGE-1), a measure of growth hormone deficiency, was 82.4 (45) micrograms/l. Lean body mass calculated by measuring total body potassium was 50 (9) kg. All patients had a normal left ventricular mass index and a normal left ventricular ejection fraction. Eight patients had abnormal left ventricular diastolic function. There was a significant correlation between IGF-1 and left ventricular mass (r = 0.45, p less than 0.02). Lean body mass was also significantly correlated with left ventricular mass (r = 0.78, p less than 0.0001) and left ventricular diastolic function (r = -0.63, p less than 0.01). The mean exercise duration was 8.6 (3.6) minutes. There was a significant correlation between serum IGF-1 and the rate-pressure product on exercise (r = 0.47, p less than 0.01). Seven patients had planar ST segment depression greater than 0.1 mV during exercise testing. In five of these patients there was rapid resolution of ST segment depression immediately after exercise. Two patients developed considerable ST segment depression, and subsequent coronary angiography showed normal coronary arteries. Exercise-induced ST segment depression was not related to the severity or duration of growth hormone deficiency or serum cholesterol concentration. CONCLUSIONS: This study suggests that left ventricular mass and the rate-pressure product are related to the degree of growth hormone deficiency, that left ventricular diastolic dysfunction is frequently seen in hypopituitarism, and that these patients may have ischaemic-like ST segment changes during exercise testing. These findings may explain the increased cardiovascular mortality in patients with hypopituitarism and may also have implications for growth hormone replacement therapy in adults.

The role of nutrition and insulin in the regulation of sex hormone binding globulin.

In an analysis of 263 women with polycystic ovary syndrome (PCOS), 91 (35%) of whom were obese (body mass index greater than 25 kg/m2), it was found that obese women with PCOS were more likely to be anovulatory and had a higher prevalence of hirsutism than the non-obese subgroup. Although serum concentrations of gonadotrophins, androstenedione and total testosterone were similar in obese and lean women with PCO, sex hormone binding globulin (SHBG) levels were significantly lower, and free testosterone correspondingly higher, in obese women. Serum concentrations of SHBG were inversely correlated with those of both fasting and glucose-stimulated insulin. A short-term, very-low-calorie diet resulted in a 2-fold increase in SHBG which was mirrored by a fall in serum insulin. Similar biochemical changes were also observed during a long-term (6-7 months) 1000 kcal diet and were associated with an improvement of menstrual function and fertility. This encourages the view that calorie restriction has an important part to play in the management of obese women with PCOS.

Correlation of plasma insulin and insulin-like growth factor-I with indices of androgen transport and metabolism in women with polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is said to be associated with hyperinsulinaemia. Insulin stimulates androgen production by ovarian tissue in vitro and previous studies have identified a positive correlation of insulin with androstenedione. The aim of the present study was to discover whether insulin levels correlate with clinical presentation and with markers of androgen transport and metabolism in women with PCOS. DESIGN: Within-group analysis of clinical and biochemical characteristics of a consecutive series of women with PCOS, focusing on correlations of plasma insulin with clinical presentation and androgens. Insulin levels were also compared with a control group of normal women. PATIENTS: Forty-seven women who presented with hirsutism, cycle abnormalities or both, with ultrasound proven PCOS, were recruited. Mean age was 26.6 +/- 0.7 years (mean +/- SEM), BMI 27.3 +/- 1.2 kg/m2. MEASUREMENTS: Plasma insulin levels were measured at 30-minute intervals for 3 hours following a 75 g glucose load. Blood was also taken for measurement of testosterone (T), androstenedione (A), free testosterone (fT), sex hormone binding globulin (SHBG) and insulin-like growth factor-I (IGF-I). Androsterone glucoronide (AG), a marker of peripheral androgen metabolism, was also measured. RESULTS: Neither basal insulin nor the sum of insulin measurements during the glucose tolerance test (sumINS) in women with PCOS were significantly different from a control group with normal ovaries. Within the PCOS group, basal insulin was greater in women with irregular cycles or amenorrhoea than in those with regular ovulatory menses (8.0 +/- 1.1 vs 3.1 +/- 1.5 mU/l, P less than 0.01) despite similarly raised androgen levels. Both basal insulin and sumINS correlated with BMI in women with PCO (r = 0.37, P less than 0.05 and r = 0.64, P less than 0.01 respectively) but not in controls. There was no significant correlation between insulin or IGF-I levels and T, A or AG despite a positive correlation of AG (but no other androgen) with BMI. SHBG showed an inverse correlation and fT correlated positively with sumINS (r = -0.51, P less than 0.01; r = 0.39, P less than 0.05). Regression analysis of each of the androgens on the other variables demonstrated no significant relationship between insulin and androgens. CONCLUSIONS: These data suggest that, in vivo, the major effect of insulin on androgen secretion is mediated by changes in SHBG rather than by direct stimulation of ovarian androgen production. Higher insulin concentrations in anovulatory compared with ovulatory women with hyperandrogenaemia may indicate that insulin resistance in the ovary contributes to the mechanism of anovulation in PCOS.

Growth hormone and the microvascular complications of diabetes.

The basis for the hypothesis that growth hormone is a permissive factor in the pathogenesis of diabetic microvascular complications is a weak one. The best way forward in this research will be to devise a pharmacological method of suppressing growth hormone secretion in diabetic subjects.

Differences in clinical and endocrine features between obese and non-obese subjects with polycystic ovary syndrome: an analysis of 263 consecutive cases.

Two hundred and sixty-three women with ultrasound-diagnosed polycystic ovary syndrome were studied of whom 91 (35%) were obese (BMI greater than 25 kg/m2). Obese women with PCOS had a greater prevalence of hirsutism (73% compared with 56%) and menstrual disorders than non-obese subjects. Total testosterone and androstenedione concentrations in serum were similar in the two subgroups but SHBG concentrations were significantly lower, and free testosterone levels higher, in obese compared with lean subjects. In addition, concentrations of androsterone glucuronide, a marker of peripheral 5 alpha-reductase activity, were higher in obese than in non-obese women with PCOS. There were no significant correlations of either SHBG or free testosterone with androsterone glucuronide suggesting that obesity has independent effects on transport and on metabolism of androgen. There were no significant differences between the subgroups in either baseline gonadotrophin concentrations or the pulsatile pattern of LH and FSH secretion studied over an 8-h period. There was, however, an inverse correlation of FSH with BMI, but only in the obese subgroup. In conclusion, the increased frequency of hirsutism in obese compared with lean women with PCOS is associated with increased bio-availability of androgens to peripheral tissues and enhanced activity of 5 alpha-reductase in obese subjects. The mechanism underlying the higher prevalence of anovulation in obese women remains unexplained.

A two-year follow-up study of serum insulinlike growth factor-I in diabetics with retinopathy.

Serum insulinlike growth factor-I (IGF-I) concentration was evaluated prospectively over two years in 35 diabetic patients with severe background or preproliferative retinopathy (group 1) and 24 diabetics with mild background retinopathy matched for age, sex, and glycemic control (group 2). In addition, 12 normal subjects were also studied to assess the variability of individual serum IGF-I levels over two years. Mean serum IGF-I (+/- SD) micrograms/l at entry, one year, and two years was not significantly different in the patient groups (157 +/- 71 v 168 +/- 77; 166 +/- 78 v 159 +/- 87; 143 +/- 58 v 159 +/- 67) or when compared with the normal subjects (181 +/- 47, 188 +/- 30; 221 +/- 56). Eight patients in the preproliferative group and none in the mild background group developed proliferative retinopathy. In this subgroup developing retinal neovascularization, serum IGF-I at the time of the first appearance of retinal new vessels was significantly higher than 3 months (1 to 4 months) before the onset of proliferation (271 +/- 94 v 196 +/- 58; P = .036). Values at the time of proliferation were not, however, significantly different from the mean serum IGF-I value of all patients in group 1 and by 4 months (3 to 6 m) had returned to their previous values. Although a transient elevation of IGF-I occurs at the time of retinal new vessel formation, the rise in serum concentration is not sufficiently great or early enough to be of clinical value as a predictor of retinal neovascularization.