Plasma steroid profiling and response to trophins to illustrate intra-adrenal dynamics.

The importance of corticosteroids in cardiovascular and other chronic disease is recognised. In addition, plasma steroid precursor-to-product ratios are useful and convenient indirect indicators of efficiency of key steroidogenic enzymes (aldosterone synthase, 11ß-hydroxylase and 17α-hydroxylase). The use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) has enabled measurement of numerous corticosteroid compounds simultaneously. However, normal responses to trophins and variation in salt intake are not well described. This study examined these parameters in a large group of healthy volunteers. Sixty normotensive volunteers were recruited and underwent infusion of angiotensin II (AngII) and ACTH, following low- and high-salt diet. Measurement of plasma steroids at baseline and 30 min after infusion of trophin was carried out by LC-MS. As expected, plasma mineralocorticoid levels increased in response to salt restriction and were suppressed with salt loading; ACTH infusion increased all corticosteroids, while AngII increased mineralocorticoids and suppressed glucocorticoid production. ACTH increased S:F but decreased DOC:B, thus the S:F ratio is a more appropriate index of 11ß-hydroxylase efficiency. The B:F ratio increased following ACTH treatment and salt restriction. A larger proportion of plasma B than generally accepted may be derived from the zona glomerulosa and this ratio may be most informative of 17α-hydroxylase activity in salt-replete subjects. Although DOC:aldosterone, B:aldosterone and 18-hydroxyB:aldosterone should provide indices of aldosterone synthase efficiency, responses of individual compounds to trophins suggest that none of them accurately reflect this. Based on these data, aldosterone synthase activity is most accurately reflected by aldosterone concentration alone.

AMP-activated protein kinase is activated in adipose tissue of individuals with type 2 diabetes treated with metformin: a randomised glycaemia-controlled crossover study.

AIMS/HYPOTHESIS: The hypoglycaemic actions of metformin have been proposed to be mediated by hepatic AMP-activated protein kinase (AMPK). As the effects of metformin and the role of AMPK in adipose tissue remain poorly characterised, we examined the effect of metformin on AMPK activity in adipose tissue of individuals with type 2 diabetes in a randomised glycaemia-controlled crossover study. METHODS: Twenty men with type 2 diabetes (aged 50-70 years) treated with diet, metformin or sulfonylurea alone were recruited from North Glasgow University National Health Service Trusts' diabetes clinics and randomised to either metformin or gliclazide for 10 weeks. Randomisation codes, generated by computer, were put into sealed envelopes and stored by the hospital pharmacist. Medication bottles were numbered, and allocation was done in sequence. The participants and investigators were blinded to group assignment. At the end of each phase of therapy adipose biopsy, AMPK activity (primary endpoint) and levels of lipid metabolism and signalling proteins were assessed. In parallel, the effect of metformin on AMPK and insulin-signalling pathways was investigated in 3T3-L1 adipocytes. RESULTS: Ten participants were initially randomised to metformin and subsequently crossed over to gliclazide, while ten participants were initially randomised to gliclazide and subsequently crossed over to metformin. No participants discontinued the intervention and the adipose tissue AMPK activity was analysed in all 20 participants. There were no adverse events or side effects in the study group. Adipose AMPK activity was increased following metformin compared with gliclazide therapy (0.057 ± 0.007 vs 0.030 ± 0.005 [mean ± SEM] nmol min(-1) [mg lysate](-1); p < 0.005), independent of AMPK level, glycaemia or plasma adiponectin concentrations. The increase was associated with reduced levels of acetyl-CoA carboxylase (ACC) protein and increased ACC Ser80 phosphorylation. In 3T3-L1 adipocytes, metformin reduced levels of ACC protein and stimulated phosphorylation of AMPK Thr172 and hormone-sensitive lipase Ser565. CONCLUSIONS: These results provide the first evidence that metformin activates AMPK and reduces ACC protein levels in human adipose tissue in vivo. Future studies are required to assess the role of adipose AMPK activation in the pharmacological effects of metformin. TRIAL REGISTRATION: ISRCTN51336867.

A screening procedure for primary aldosteronism based on the Diasorin Liaison automated chemiluminescent immunoassay for direct renin.

BACKGROUND: Primary aldosteronism (PA), the most common secondary cause of hypertension, can be screened for using the aldosterone/renin ratio. This ratio is raised in PA and its accuracy depends on the ability to measure plasma renin at extremely low concentrations. METHODS: We compared two different procedures for assessing plasma renin. The conventional method, which measures plasma renin activity (PRA), is technically demanding and laborious, and the Diasorin Liaison method, which measures plasma renin concentration (PRC), is an automated immunoassay. Results from each method were used to calculate the aldosterone/renin ratio (ARR) and the performance of the Diasorin Liaison method compared with that of the conventional assay using receiver operator characteristic curves. RESULTS: The analytical and functional sensitivity of the PRC method were 2.1 and 5 microIU/mL, respectively. Intra- and inter-assay precision were <7.2% and 10.4%, respectively. There was significant (9%) prorenin interference. Samples with PRA > 1.0 ng/mL/h showed significant correlation with PRC (r = 0.93; P < 0.05; n = 146); however, with PRA < 1.0 ng/mL/h, no significant correlation occurred (r = 0.14; P < 0.05; n = 79). An aldosterone (pmol/L)/PRC(microIU/mL) ratio of >35, in patients with aldosterone >300 pmol/L, resulted in 100% sensitivity and 93% specificity, when compared with the commonly accepted aldosterone (pmol/L)/PRA (ng/mL/h) ratio of >750, in identifying patients who may suffer from PA. CONCLUSION: This study indicates the feasibility of using the automated PRC assay as a replacement for the conventional manual PRA assay in calculating the ARR as a first-line screen for PA.

Aldosterone status associated with insulin resistance in patients with heart failure--data from the ALOFT study.

BACKGROUND: Aldosterone has a key role in the pathophysiology of heart failure. In around 50% of such patients, aldosterone "escapes" from inhibition by drugs that interrupt the renin-angiotensin axis; such patients have a worse clinical outcome. Insulin resistance is a risk factor in heart failure and cardiovascular disease. The relation between aldosterone status and insulin sensitivity was investigated in a cohort of heart failure patients. METHODS: 302 patients with New York Heart Association (NYHA) class II-IV heart failure on conventional therapy were randomised in the ALiskiren Observation of heart Failure Treatment study (ALOFT), designed to test the safety of a directly acting renin inhibitor. Plasma aldosterone and 24-hour urinary aldosterone excretion, as well as fasting insulin and homeostasis model assessment of insulin resistance (HOMA-IR) were measured. Subjects with aldosterone escape and high urinary aldosterone were identified according to previously accepted definitions. RESULTS: 20% of subjects demonstrated aldosterone escape and 34% had high urinary aldosterone levels. At baseline, there was a positive correlation between fasting insulin and plasma (r = 0.22 p<0.01) and urinary aldosterone(r = 0.19 p<0.03). Aldosterone escape and high urinary aldosterone subjects both demonstrated higher levels of fasting insulin (p<0.008, p<0.03), HOMA-IR (p<0.06, p<0.03) and insulin-glucose ratios (p<0.006, p<0.06) when compared to low aldosterone counterparts. All associations remained significant when adjusted for potential confounders. CONCLUSIONS: This study demonstrates a novel direct relation between aldosterone status and insulin resistance in heart failure. This observation merits further study and may identify an additional mechanism that contributes to the adverse clinical outcome associated with aldosterone escape.

Effects of genetic variation in the aldosterone synthase (CYP11B2) gene on enzyme function.

OBJECTIVE: Evidence suggests that high levels of aldosterone lead to hypertension and increased risk of cardiovascular disease. Around 15% of patients with essential hypertension have a raised aldosterone to renin ratio (ARR) suggesting that aldosterone production is inappropriately high in relation to its principal agonist angiotensin II. This may be due to increased activity of aldosterone synthase caused by genetic variation in the CYP11B2 gene. We screened the coding region of human CYP11B2 for genetic variants and tested their effects on function in vitro. PROTOCOL: Normotensive subjects (n = 69) were screened for sequence variants in the coding region of CYP11B2 by single-stranded conformation polymorphism (SSCP) analysis and sequencing. The effects of nonsynonymous variants on enzyme activity were assessed in JEG-3 cells transiently transfected with wild-type or variant expression plasmids. The conversion of the substrate 11-deoxycorticosterone (DOC) to corticosterone (B) and aldosterone was measured. RESULTS: Twenty variants were detected in CYP11B2 and eight analysed functionally (Arg87Gly, Asn281Thr, Gly288Ser, Lys296Asn, Asp335Asn, Gln404Arg, Ala414Pro and His439Tyr). Corticosterone synthesis was unaltered and aldosterone synthesis reduced in variant Arg87Gly; Asn281Thr increased corticosterone and decreased aldosterone production; Gly288Ser increased corticosterone production and abolished aldosterone production; Lys296Asn reduced both corticosterone and aldosterone production; Asp335Asn increased corticosterone synthesis but did not affect aldosterone production. Variants Gln404Arg, Ala414Pro and His439Tyr showed increases in both corticosterone and aldosterone synthesis compared to the wild-type. CONCLUSION: The study confirms the genetic variability of the CYP11B2 gene and provides us with additional valuable structure-function information.

Effect of variation in CYP11B1 and CYP11B2 on corticosteroid phenotype and hypothalamic-pituitary-adrenal axis activity in hypertensive and normotensive subjects.

BACKGROUND: Aldosterone is important in the development of hypertension. We have shown that a single nucleotide polymorphism (SNP) (-344T) in the 5' regulatory region (UTR) of the gene encoding aldosterone synthase (CYP11B2) associates with aldosterone excess and hypertension as well as altered adrenal 11-hydroxylation efficiency (deoxycortisol to cortisol). This conversion is carried out by the enzyme 11beta-hydroxylase, encoded by the adjacent gene, CYP11B1. We proposed that the effects of CYP11B2 are explained by linkage disequilibrium (LD) across the CYP11B locus. We have demonstrated high LD across this locus and identified two SNPs in the 5' UTR of CYP11B1 (-1859 G/T, -1889 A/G) that associate with reduced transcription in vitro and altered 11-hydroxylation efficiency in vivo. Accordingly, we hypothesized that the reduced adrenal 11-hydroxylation may lead to chronic resetting of the pituitary-adrenal axis, with chronically increased ACTH drive resulting in aldosterone excess. METHODS: To test this, we examined hypothalamic-pituitary-adrenal (HPA) axis activity in hypertensive and normotensive individuals stratified according to genotype at CYP11B2 (-344T/C) and CYP11B1 (-1859 G/T, -1889 A/G). Fifty-six subjects homozygous for CYP11B2 SNP (27 TT, 12 CC), and 38 homozygous for CYP11B1 SNPs (18 TTGG, 20 GGAA) were recruited. Diurnal variation and the effects of dexamethasone suppression and ACTH stimulation on plasma aldosterone, cortisol and ACTH under controlled conditions were studied. RESULTS: Subjects with SNPs associated with reduced 11-hydroxylation efficiency (-344T CYP11B2; TTGG CYP11B1) showed reduced inhibition of ACTH after dexamethasone (P = 0.05) and an altered cortisol-ACTH relationship (decreased cortisol-ACTH ratio, P < 0.02). The same individuals also demonstrated close correlations between plasma cortisol and aldosterone (-344T CYP11B2 r = 0.508, P < 0.004; TTGG CYP11B1 r = 0.563, P < 0.003) suggesting that there was common regulation (possibly ACTH) of these hormones in genetically susceptible subjects. CONCLUSIONS: Variation in CYP11B2 and CYP11B1 associates with chronic up-regulation of the HPA axis. These novel data support the suggestion that chronic aldosterone excess, in genetically susceptible individuals, may be a consequence of increased ACTH drive to the adrenal and identify novel molecular mechanisms that may lead to the development of hypertension within the general population.

Hypertension.

Hypertension is a common condition affecting one in four adults. It is a leading cause of cardiovascular morbidity and mortality and appropriate treatment strategies that are both clinically and cost effective are key to the management of this condition. Recent guidelines have focused on addressing total cardiovascular risk and recommended rational combinations of antihypertensives as well as highlighting the importance of lifestyle intervention. Recent advances have increased understanding of the pathogenesis of hypertension and have opened the possibility of novel interventions for treatment.

Phenotypic consequences of variation across the aldosterone synthase and 11-beta hydroxylase locus in a hypertensive cohort: data from the MRC BRIGHT Study.

BACKGROUND: Aldosterone is an important cardiovascular hormone; 15% of hypertensive subjects have alteration in aldosterone regulation, defined by a raised ratio of aldosterone to renin (ARR). Studies of the aldosterone synthase gene (CYP11B2) have focused on a single nucleotide polymorphism in the 5'promoter region (-344 C/T). In normotensive subjects, the T allele associates with raised levels of the 11-deoxysteroids, deoxycorticosterone and 11-deoxycortisol which are substrates for 11beta-hydroxylase, encoded by the adjacent and homologous gene, CYP11B1. We have speculated that this altered 11beta-hydroxylase efficiency leads to increased ACTH drive to the adrenal gland to maintain cortisol production and reported herein the association between the -344 C/T single nucleotide polymorphism (SNP) and adrenal steroid production in subjects with essential hypertension. METHODS: The CYP11B2-344 C/T polymorphism was genotyped and urinary excretion of adrenal steroid metabolites was measured (by GCMS) in 511 unrelated hypertensives from the Medical Research Council (MRC) British Genetics of Hypertension (BRIGHT) study. RESULTS: Thirty-five per cent of subjects were homozygous for the -344T allele whilst 16% were CC homozygotes. There was no difference in cortisol excretion rate between the two genotype groups but the index of adrenal 11beta-hydroxylation (ratio of tetrahydrodeoxycortisol/total cortisol) was significantly higher in the TT group (P < 0.005) than in the CC group. Excretion rates of the major urinary metabolite of aldosterone (tetrahydroaldosterone) correlated strongly with the ACTH-regulated steroids, cortisol (r = 0.437, P < 0.0001) and total androgen metabolites (r = 0.4, P < 0.0001) in TT but not CC subjects. CONCLUSIONS: Hypertensives homozygous for the -344 T allele of CYP11B2 demonstrate altered 11beta-hydroxylase efficiency (CYP11B1); this is consistent with the hypothesis of a genetically determined increase in adrenal ACTH drive in these subjects. The correlation between excretion of aldosterone and cortisol metabolites and suggests that, in TT subjects, ACTH exerts an important common regulatory influence on adrenal corticosteroid production in subjects with hypertension.

Endothelium-dependent relaxation is resistant to inhibition of nitric oxide synthesis, but sensitive to blockade of calcium-activated potassium channels in essential hypertension.

In human essential hypertension (EH), endothelium-dependent relaxation can occur independent of nitric oxide (NO) and prostacyclin (PGI(2)). Recent in vivo data suggest that rapid compensatory upregulation of endothelial cytochrome P450 epoxygenase 2C9 occurs to preserve vasorelaxation under conditions of decreased NO bioavailability. As one of the vascular actions of CYP2C9 is to modulate small and intermediate conductance endothelial calcium-activated potassium channels (SK(Ca) and IK(Ca)), we examined whether endothelium-dependent relaxation is sensitive to inhibitors of these channels (apamin and charybdotoxin) in resistance-sized vessels from human with EH. Subcutaneous gluteal biopsies were performed on 12 humans with EH and 12 matched control subjects. Resistance arteries were dissected and relaxation responses to carbachol were assessed ex vivo using wire myography in the presence of: (i) N(G)-nitro-L-arginine (L-NOARG)/indomethacin; and (ii) apamin/charybdotoxin. Maximal carbachol relaxation was impaired in EH vs control subjects. No differences in responses were observed with the endothelium-independent agonist, S-nitroso-N-acetyl-penicillamine. Relaxation to carbachol was attenuated following incubation with L-NOARG/indomethacin in vessels from control subjects (P<0.01 analysis of variance (ANOVA)), but not in vessels from patients with EH. The reverse pattern was seen following apamin/charybdotoxin with carbachol relaxation attenuated only in EH vessels (P<0.001 ANOVA). Endothelium-dependent relaxation is resistant to endothelial nitric oxide synthase inhibition but sensitive to blockade of calcium-activated potassium channels in human EH. Studies with more specific inhibitors are required to determine whether this response is mediated by endothelial potassium channel subtypes (SK(Ca) and IK(Ca)).

Variability in hydrocortisone plasma and saliva pharmacokinetics following intravenous and oral administration to patients with adrenal insufficiency.

OBJECTIVE: The best method for determining hydrocortisone replacement therapy is not well defined. This study aimed to assess interindividual variability in cortisol pharmacokinetics and to investigate whether measurement of salivary cortisol provides a useful alternative to plasma concentration measurements. DESIGN: Intravenous (IV) and oral crossover. PATIENTS: Twenty-seven patients with primary or secondary adrenal insufficiency who had been on stable replacement therapy for at least 3 months. MEASUREMENTS: Plasma and salivary concentrations of cortisol were measured up to 8 h following administration of hydrocortisone. RESULTS: After IV administration, Cmax ranged from 715 to 8313 nmol/l, area under the curve (AUC) from 1112 to 12 177 nmol h/l and cortisol clearance had a median (range) of 0.267 (0.076-0.540) l/h/kg. After oral administration, Cmax ranged from 422 to 1554 nmol/l, AUC 1081-5471 nmol h/l and oral clearance had a median (range) of 0.267 (0.081-0.363) l/h/kg. There was no clear relationship between paired saliva and plasma cortisol concentrations after IV or oral dosing. Plasma and salivary AUC(2-8 h) after IV administration were highly correlated (r2 = 0.77) but differences between predicted and measured plasma AUCs ranged from 3% to 90%. There was a poor correlation between plasma and saliva AUC(2-6 h) after oral administration (r2 = 0.16). CONCLUSIONS: The wide interindividual variability in plasma and salivary profiles of cortisol following the administration of IV and oral hydrocortisone to patients with adrenal insufficiency and the poor correlation between salivary and plasma measurements suggest that salivary cortisol measurements cannot be used for individual hydrocortisone dosage adjustment.

Endogenous corticosteroid biosynthesis in subjects after bilateral adrenalectomy.

OBJECTIVE: Corticosteroids can be synthesized in extra-adrenal tissues but the contribution of this to circulating levels in humans is not known. Previous in vitro studies suggest that the 'hybrid' corticosteroid 18-oxocortisol (18-oxoF) is produced from cortisol by aldosterone synthase. We looked for evidence of extra-adrenal production of this and other corticosteroids in 10 subjects stable on long-term glucocorticoid replacement following bilateral adrenalectomy. METHODS: In phase 1, patients were maintained on cortisol alone (30 mg/day), in phase 2 dexamethasone (2 mg/day), and in phase 3, both cortisol and dexamethasone. Each phase lasted 3 days. MEASUREMENTS: On the last day of each phase, 24-h urine collection was performed for analysis of steroid metabolite excretion [using gas chromatography-mass spectrometry (GCMS)] and plasma aldosterone and renin were measured (by radioimmunoassay). RESULTS: Cortisol metabolite excretion rate [tetrahydrocortisone (THE) + tetrahydrocortisol (THF) + allotetrahydrocortisol (aTHF)] fell from 9169 nmol/24 h in phase 1 to 22 nmol/24 h in phase 2, rising to 6843 nmol/24 h in phase 3. Tetrahydroaldosterone (THAldo) excretion was readily detectable and did not alter significantly between phases (26.5, 23.5 and 28.5 nmol/24 h, respectively; P = 0.474). 18-Hydroxycortisol (18-OHF) excretion was easily detectable in phases 1 and 3 (252.5 and 212 nmol/24 h), falling in phase 2 (12 nmol/24 h). 18-oxoF excretion rates were lower but followed a similar pattern (1.62, 0.085 and 1.785 nmol/24 h in phases 1, 2 and 3, respectively). CONCLUSIONS: Significant levels of adrenal steroids are found in adrenalectomized subjects. We speculate that this occurs at extra-adrenal sites or in residual adrenal cortex tissue in an ACTH-independent manner. Our data suggest that aldosterone synthase, acting on cortisol, is the source of 18-oxoF and 18-OHF in these subjects. Further studies of corticosteroid production within adrenalectomized subjects, looking for evidence of adrenal regrowth or residual adrenal tissue, are justified.

Effects of low-dose continuous combined hormone replacement therapy on glucose homeostasis and markers of cardiovascular risk in women with type 2 diabetes.

BACKGROUND: Low-dose hormone replacement therapy (HRT) has attracted interest for the treatment of postmenopausal symptoms in diabetes because of concerns about increased risk of coronary heart disease (CHD) and stroke with conventional HRT containing conjugated equine oestrogens (CEEs) and medroxyprogesterone acetate (MPA). OBJECTIVES: We assessed the effects on glucose homeostasis and cardiovascular risk factors of continuous oral 17beta oestradiol (1 mg) and norethisterone (0.5 mg) in postmenopausal women with type 2 diabetes. DESIGN: Double-blind, randomized placebo-controlled trial. ASSESSMENTS: Hyperinsulinaemic isoglycaemic clamp and cardiovascular risk factors were assessed before and after 3 months of treatment. RESULTS: Twenty-eight women completed the study. HRT decreased fasting glucose compared with placebo [-9.4% with HRT vs.+2.3% for placebo, 95% confidence interval (CI) -23.2 to -0.3] and total cholesterol (-13.7 vs.+1.0%, 95% CI -22.4 to -3.1%) No significant effect was seen on metabolic clearance rate of glucose, glycated haemoglobin (HbA1c), triglycerides, high density lipoprotein (HDL)-cholesterol or C-reactive protein (CRP). CONCLUSIONS: In women with type 2 diabetes, low-dose HRT decreased fasting glucose and total cholesterol without detectable adverse effects on glucose clearance, triglycerides and CRP as reported with conventional HRT.

The link between abdominal obesity, metabolic syndrome and cardiovascular disease.

AIM: The prevalence of metabolic syndrome has increased dramatically in recent years, and the cluster of metabolic abnormalities it encompasses results in increased cardiovascular morbidity and mortality. The role of abdominal (visceral) obesity and the underlying molecular and cellular mechanisms central to this association have been the subject of intensive research in recent times. The aim of this review is to correlate data in this area, highlighting the central role of excess visceral fat and its secreted adipokines, and to review existing and emerging therapies. DATA SYNTHESIS: Data were generated from a search of the PubMed database using the terms 'abdominal obesity', 'metabolic syndrome', 'insulin resistance', 'adipokines', 'interleukin-6 (IL-6)', 'adiponectin', 'tumour necrosis factor-alpha (TNF-alpha)' and 'cardiovascular disease'. CONCLUSION: Metabolic syndrome is associated with a pro-inflammatory state, and the role of visceral obesity is thought to be central to this. Visceral obesity leads to alteration of the normal physiological balance of adipokines, insulin resistance, endothelial dysfunction and a pro-atherogenic state. In association with this, the presence of conventional cardiovascular risk factors such as hypertension, dyslipidaemia and smoking results in a significantly elevated cardiovascular and metabolic (cardiometabolic) risk. Better understanding of the molecular mechanisms central to this association has led to the development of potential therapeutic agents.

Blunted response to systemic nitric oxide synthase inhibition in the cerebral circulation of patients with Type 2 diabetes.

AIMS: Diabetes is a major risk factor for stroke, but the mechanisms that impart the excess risk are unclear. Endothelial dysfunction, which has been demonstrated in the coronary and peripheral vasculature of diabetic patients, is an important early marker of vascular disease. However, the effect of diabetes on cerebrovascular endothelium has not been examined. We sought to investigate the effect of diabetes on basal cerebrovascular endothelial function as assessed by response to the nitric oxide synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA). METHODS: Fourteen men with Type 2 diabetes and 15 age-matched male control subjects were recruited. The participants had no clinically evident vascular disease and were taking no vasoactive or lipid-lowering medication. Each received a single 15-min intravenous infusion of L-NMMA (0.8 mol/kg/min). Cerebral blood flow was assessed by colour Doppler imaging of the internal carotid artery (ICA) at 10-min intervals for 20 min prior to and following the infusion. Middle cerebral artery velocity (MCAv) was assessed by transtemporal Doppler ultrasound at the same time points. RESULTS: L-NMMA produced a mean reduction in ICA flow area under curve (AUC) in the control group of 12.8 +/- 17.8% compared with a 2.1 +/- 21.7% reduction in the group with diabetes (P < 0.05), indicating blunted basal cerebrovascular response to NOS inhibition in the diabetic group. There was no significant change in MCAv following L-NMMA in either group. Mean +/- sd MAP rose 6.4 +/- 4.2 mmHg in the control group vs. 8.8 +/- 3.5 mmHg in the diabetic group [P = not significant (NS)]. No adverse event or symptom was reported. CONCLUSIONS: Response to NOS inhibition is impaired in the cerebral circulation of patients with diabetes. This observation is consistent with the elevated cerebrovascular risk reported in this population, and may represent a future therapeutic target in stroke prevention.

The aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) genes are not expressed in the rat heart.

Aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) catalyze the production of aldosterone and corticosterone, respectively, in the rat adrenal cortex. Recently, there has been some debate as to whether these corticosteroids are also produced in the hearts of rodents and humans, possibly contributing to the development of hypertrophy and myocardial fibrosis. To investigate this, we have used our established, highly sensitive real-time quantitative RT-PCR method to measure CYP11B1 and CYP11B2 mRNA levels in adrenal and cardiac tissue from several rat models of cardiovascular pathology. We have also studied isolated adult rat ventricular myocytes treated with angiotensin II and ACTH. Total RNA was isolated from the adrenal and cardiac tissue of 1) male Wistar rats with heart failure induced by coronary artery ligation and sham-operated controls; 2) stroke-prone spontaneously hypertensive rats and Wistar Kyoto rats as controls; 3) cyp1a1Ren-2 transgenic rats and Fischer controls; 4) isolated adult Sprague-Dawley ventricular myocytes incubated with 11-deoxycorticosterone (DOC), DOC plus angiotensin II, or DOC plus ACTH. Adrenal CYP11B2 expression was significantly increased in transgenic rats compared with Fischer controls (1.3 x 10(9)+/- 1.2 x 10(9) vs. 2.1 x 10(7) +/- 7.0 x 10(6) copies/microg RNA; P < 0.05). There were no other significant differences in adrenal CYP11B2 or CYP11B1 expression between the model animals and their respective controls. Cardiac CYP11B1 and CYP11B2 mRNA transcript levels from all in vivo and in vitro groups were never greater than 100 copies per microgram total RNA and therefore too low to be detected reproducibly. This suggests that cardiac corticosteroid production is unlikely to be of any physiological or pathological significance.

A rare variant of the leptin gene has large effects on blood pressure and carotid intima-medial thickness: a study of 1428 individuals in 248 families.

BACKGROUND: Rare mutations in the leptin (LEP) gene cause severe obesity. Common polymorphisms of LEP have been associated with obesity, but their association with cardiovascular disease has been little studied. We have examined the impact of both common and rare polymorphisms of the LEP gene on blood pressure (BP), subclinical atherosclerosis as measured by carotid intima-medial thickness (CIMT), and body mass index (BMI) in a large family study. METHODS: Five polymorphisms spanning LEP were typed in 1428 individuals from 248 nuclear families. BP, CIMT, BMI, and plasma leptin were measured. RESULTS: The polymorphisms typed captured all common haplotypes present at LEP. There was strong association between a rare polymorphism in the 3' untranslated region of LEP (C538T) and both pulse pressure (p = 0.0001) and CIMT (p = 0.008). C/T heterozygotes had a 22% lower pulse pressure and a 17% lower CIMT than C/C homozygotes. The polymorphism accounted for 3-5% of the population variation in pulse pressure and CIMT. There was no association between any LEP polymorphism and either BMI or plasma leptin level. CONCLUSIONS: This large family study shows that the rare T allele at the C538T polymorphism of LEP substantially influences pulse pressure and CIMT, but does not appear to exert this effect through actions on plasma leptin level or BMI. This suggests that autocrine or paracrine effects in vascular tissue may be important physiological functions of leptin. This study also provides evidence that rare polymorphisms of particular genes may have substantial effects within the normal range of certain quantitative traits.

Reduced insulin-stimulated GLUT4 bioavailability in stroke-prone spontaneously hypertensive rats.

AIMS/HYPOTHESIS: Insulin-stimulated glucose transport is impaired in a genetic model of hypertension, the stroke-prone spontaneously hypertensive rat (SHRSP), yet the molecular mechanisms that underlie this defect in the animals remain unclear. METHODS: We examined the effects of insulin on the trafficking of the insulin-responsive glucose transporter GLUT4 to the plasma membrane in isolated adipocytes from SHRSP and normotensive control Wistar-Kyoto (WKY) rats. RESULTS: Treatment of isolated adipocytes with insulin resulted in trafficking of GLUT4 to the plasma membrane. There was no significant difference in the magnitude of insulin-stimulated GLUT4 trafficking from intracellular membranes to the plasma membrane between strains. In contrast, we demonstrated that there is a significant reduction in GLUT4 accessible to the glucose photolabel Bio-LC-ATB-BGPA at the plasma membrane of SHRSP adipocytes compared with control rats. CONCLUSIONS/INTERPRETATION: We propose that a large proportion of GLUT4 translocated to the plasma membrane in response to insulin is not able to bind substrate and catalyse transport in the SHRSP. Therefore, there is a reduction in bioavailable GLUT4 in SHRSP animals that is likely to account, at least in part, for the reduced insulin-stimulated glucose uptake.

Glucocorticoids contribute to the heritability of leptin in Scottish adult female twins.

OBJECTIVE: The precise interactions between glucocorticoids and leptin are complex and poorly understood. The aim of the study was to investigate whether the glucocorticoid/leptin interaction is influenced by shared environmental or genetic factors. DESIGN: We investigated the heritability of body mass index (BMI), circulating leptin and urinary glucocorticoid metabolites [tetrahydrocortisol (THF), alloTHF and tetrahydrocortisone (THE)] in 54 monozygotic (MZ) and 39 dizygotic (DZ) female twins. Analysis was performed using a structural equation modelling package Mx, developed by Neale. RESULTS: Leptin and BMI showed substantial heritability (68.3% and 71.3%, respectively). Bivariate analysis indicated that the genetic determinants of BMI and leptin are partly shared. Total cortisol metabolites (THF + alloTHF + THE), the (THE + alloTHF)/THE ratio [a marker of 11beta-hydroxysteroid dehydrogenase (11HSD) activity] and the alloTHF/THF ratio (marker for 5alpha-reductase activity) followed an environmental pattern. The heritability of leptin was significantly lowered to 63.8% (P = 0.012) when values were corrected for the influence of total cortisol metabolites but unaffected by markers of 11HSD and 5alpha-reductase activity. CONCLUSIONS: We confirm that the genetic influence on both BMI and the circulating leptin concentration is substantial and show that these genetic determinants are highly correlated. These genetic factors, which are more likely to be dominant than additive, can be modestly but significantly modified by urinary total cortisol metabolites implying an adrenal influence.

Higher carotid-radial pulse wave velocity in healthy offspring of patients with Type 2 diabetes.

AIMS: To determine whether carotid-radial pulse wave velocity (crPWV), a simple non-invasive measurement of muscular artery structure and function, is increased in offspring of patients with Type 2 diabetes compared with well-matched controls with no family history of diabetes. Serum levels of intercellular adhesion molecule-1 (sICAM-1) were also examined. METHODS: Offspring (n = 19, M = 8) were recruited via contact with patients attending clinics. Controls (n = 19, M = 8) were recruited by advertisement. crPWV was measured using COMPLIOR. Blood pressure and heart rate were determined and fasting blood taken for measurement of metabolic and endothelial parameters. RESULTS: Offspring and controls were well matched [mean (sd)] for age [33.1 (9.6) vs. 32.8 (9.5) years], body mass index [24.8 (4.9) vs. 24.3 (3.4) kg/m2], waist circumference [78.3 (2.3) vs. 76.3 (2.5) cm], and systolic blood pressure [120 (9.3) vs. 119 (14.2) mmHg]. crPWV was 10% higher in the offspring [9.94 (1.3) m/s] compared with controls [9.01 (1.2) m/s, P = 0.02] despite similar pulse pressure [52 (10.5) vs. 53.5 (9.3) mmHg] and resting heart rate [71 (8.7) vs. 69 (14.0) beats/min]. They also showed a trend toward higher sICAM-1 [217 (55) vs. 188 (40) ng/ml, P = 0.07] concentrations which were also strongly correlated to crPWV in offspring (r = 0.63, P = 0.004). CONCLUSIONS: Vascular dysfunction in the form of increased muscular artery stiffness is present from an early stage in subjects at higher risk of developing diabetes. This may be secondary to impaired activation of endothelial signalling pathways in the context of inherited insulin resistance.