A new test for autonomic cardiovascular and neuroendocrine responses in diabetes mellitus: evidence for early vagal dysfunction.

AIMS/HYPOTHESIS: Diabetic autonomic neuropathy affects many physiological systems, producing a variety of important clinical manifestations. It is associated with high morbidity and mortality, particularly during times of stress. This is thought to be due to an increased risk of cardiac arrhythmias, although the exact mechanisms involved have yet to be fully elucidated. The aim of the present study was to investigate the endocrine, cardiac autonomic and psychological responses of diabetic patients with and without autonomic neuropathy to a single breath of 35% CO(2). METHODS: The 35% CO(2) challenge was performed in 20 male diabetic subjects, 11 of whom had autonomic neuropathy. RESULTS: Baseline and stimulated cortisol, prolactin, systolic blood pressure and emotional arousal were similar in the two groups. However, subjects with autonomic neuropathy failed to demonstrate the expected CO(2)-induced bradycardia seen in the non-neuropathic patients (p<0.0001). CONCLUSIONS/INTERPRETATION: The CO(2) challenge can be safely and easily administered to produce hypothalamic-pituitary-adrenal axis and cardiac autonomic activation, as well as emotional arousal. The test clearly distinguishes between subjects with and without cardiac autonomic neuropathy and could be an important adjunct to the methods currently available for the investigation and diagnosis of diabetic autonomic neuropathy.

Sensitivity to sulphonylureas in patients with hepatocyte nuclear factor-1alpha gene mutations: evidence for pharmacogenetics in diabetes.

INTRODUCTION: Maturity-onset diabetes of the young (MODY) is characterized by autosomal dominantly inherited, early-onset, non-insulin-dependent diabetes. Mutations in the hepatocyte nuclear factor (HNF)-1alpha gene are the commonest cause of MODY. Individual patients with HNF-1alpha mutations have been reported as being unusually sensitive to the hypoglycaemic effects of sulphonylurea therapy. We report three patients, attending a single clinic, with HNF-1alpha mutations that show marked hypersensitivity to sulphonylureas. CASE REPORTS: In cases 1 and 2 there were marked changes in HbA1c on cessation (4.4% and 5.8%, respectively) and reintroduction (5.0% and 2.6%) of sulphonylureas. Case 3 had severe hypoglycaemic symptoms on the introduction of sulphonylureas despite poor glycaemic control and was shown with a test dose of 2.5 mg glibenclamide to have symptomatic hypoglycaemia (blood glucose 2 mmol/l) after 4 h despite eating. CONCLUSIONS: HNF-1alpha MODY diabetic subjects are more sensitive to sulphonylureas than Type 2 diabetic subjects and this is seen in different families, with different mutations and may continue up to 13 years from diagnosis. This is an example of pharmacogenetics, with the underlying aetiological genetic defect altering the pharmacological response to treatment. The present cases suggest that in HNF-1alpha MODY patients: (i) sulphonylureas can dramatically improve glycaemic control and should be considered as initial treatment for patients with poor glycaemic control on an appropriate diet; (ii) hypoglycaemia may complicate the introduction of sulphonylureas and therefore very low doses of short acting sulphonylureas should be used initially; and (iii) cessation of sulphonylureas should be undertaken cautiously as there may be marked deterioration in glycaemic control.

Physiological control of splanchnic blood flow by adrenaline: studies during acute hypoglycaemia in man.

Superior mesenteric artery blood flow (SMABF) increases significantly during and after the hypoglycaemia reaction in healthy humans. To investigate the mechanisms controlling this phenomenon, SMABF and plasma catecholamines were measured in healthy human volunteers. In 10 controls, hypoglycaemia was induced by insulin infusion (2.5 m-units.min-1.kg-1). In six subjects, beta-blockade by propranolol infusion (0.7 microgram.min-1.kg-1) preceded insulin infusion and was continued throughout the study. Following the hypoglycaemia reaction, the glucose nadir was similar in both groups. In controls, increases in SMABF [42.4+/-6.1% (mean+/-S.E.M.); P<0. 001], cardiac output (34.3+/-2.3%; P<0.001) and pulse rate (from 63. 9+/-2.7 to 82.5+/-3.1 beats/min; P<0.001) occurred. Superior mesenteric artery resistance fell by 32.4+/-3.3% (P<0.001). Under beta-blockade, decreases in SMABF (34.8+/-2.9%; P<0.001) and pulse rate (from 59.5+/-0.2 to 51.8+/-2.2 beats/min; P<0.001) occurred. Superior mesenteric artery resistance increased (peak +30.8+/-12.3%; not significant). Subjects showed greater increases in adrenaline (P<0.006) and noradrenaline (P<0.022) concentrations than controls. Mesenteric hyperaemia associated with hypoglycaemia in man appears to be mediated by a beta-adrenergic mechanism that is activated by increased circulating levels of adrenaline.

Increased splanchnic blood flow after hypoglycaemia in diabetic and normal man: evidence against glucagon as a mediator.

Superior mesenteric artery blood flow increases significantly after hypoglycaemia in healthy humans. Glucagon has vasoactive properties but its role in hypoglycaemic hyperaemia is unclear. To assess this role, we studied the superior mesenteric artery blood flow response to hypoglycaemia of patients with uncomplicated Type 1 (insulin-dependent) diabetes mellitus of at least 10 years duration; a group known to have defective glucagon response to hypoglycaemia. Hypoglycaemia was induced using an intravenous infusion of soluble human insulin (2.5 m-units.min-1.kg-1) discontinued at a plasma glucose of 2.5 mmol/l. Superior mesenteric artery blood flow was measured using transcutaneous duplex Doppler ultrasound. Plasma samples were assayed for glucose, insulin, glucagon, catecholamines, growth hormone and cortisol. Plasma glucose concentration fell to a nadir of 1.8 (0.3) mmol/l in patients and 1.4 (0.1) mmol/l in controls. Plasma glucagon concentration was unchanged in patients from a baseline level of 111.7 (13.1) ng/l but rose in controls from 105 (8.5) to a peak of 239 (3.1) ng/l (P<0.001). Superior mesenteric artery blood flow increased in both groups: from 385 (29) to 921 (100) ml/min (140% increase; P<0.05) in patients and from 517 (50) to 790 (67) (53% increase; P<0.001) in controls. This study shows that patients with Type 1 diabetes have a normal splanchnic vascular hyperaemic response to hypoglycaemia despite defective glucagon counter-regulation. These results support our previous work suggesting that glucagon is not a major mediator of this response; it seems likely that circulating adrenaline is the major regulatory mechanism.

Suspected hypoglycaemia in out patient practice: accuracy of dried blood spot analysis.

OBJECTIVE: The assay of dried blood spots on filter paper to determine blood glucose concentration has been used to detect hypoglycaemia in out patients. We assessed the accuracy of this approach in assaying blood glucose concentrations in the hypoglycaemic range. DESIGN: Volunteers were rendered hypoglycaemic by intravenous infusion of insulin. The glucose concentration in simultaneously taken blood samples was measured either fresh or after drying on filter paper. PATIENTS: Twenty-four healthy young volunteers and 9 patients with insulin-dependent diabetes were studied. MEASUREMENTS: Plasma glucose concentrations were measured using a standard auto analyser glucose oxidase method. Whole blood taken simultaneously was placed on prepared filter paper and allowed to dry; glucose concentration was then measured using a well-established technique. A correction factor was applied to convert the glucose concentration of plasma to that of whole blood. The relationship between glucose concentrations measured by the two methods was determined by regression coefficient. RESULTS: In the unequivocally hypoglycaemic range (plasma < or = 2.5 mmol/l), corrected dried blood spot glucose concentrations significantly correlated with standard plasma glucose concentrations (r = 0.81; P < 0.001). The dried blood spot method had a sensitivity of 91%. In the range designated probable hypoglycaemia (plasma < or = 3.3 mmol/l), there was also significant correlation (r = 0.90; P < 0.001) and the sensitivity was 96%. The specificity of the dried blood spot method was 100% in both ranges. CONCLUSIONS: Measurement of glucose concentrations in dried blood spots is specific and sensitive in the hypoglycaemic range. The present study indicates that hypoglycaemia may be excluded or confirmed respectively when levels in excess of 3.7 or below 2.8 mmol/l are found in uncorrected dried blood spot analysis.

The scotopic electroretinogram to blue flashes and pattern reversal visual evoked potentials in insulin dependent diabetes.

Pattern reversal visual evoked responses (PR-VEPs), the electroretinogram (ERG) to blue flashes of light in dark adaptation, the steady state ERG to 40 Hz flicker, and alterations in pupil diameter following dark adaptation were studied in 56 juvenile onset diabetics, 34 of whom had no ophthalmoscopic or photographic evidence of diabetic retinopathy (DR-group). The remaining 22 had mild background retinopathy (DR+group). Normal data was obtained from 24 subjects matched for age and sex with diabetics. Skin electrodes were used for all recordings. The scotopic 'b' wave of the ERG was significantly lower in amplitude in the DR- diabetics and it was even more reduced in the DR+group. The PR-VEP was significantly delayed in diabetics but there was no difference between the two groups. The steady state ERG was not significantly different between normals and diabetics. These findings indicate that retinal and more central abnormalities develop early in diabetics. Detection of objective electrophysiological abnormalities may be used to identify persons at risk of developing retinopathy and to monitor the effects of treatment.

Decreased sialidase activity in mononuclear leucocytes of type 1 diabetic subjects: relationship to diabetic complications and glycaemic control.

Leucocyte surface sialic acid content influences surface charge, deformability, and leucocyte-endothelial interaction. Abnormal leucocyte structure and function contributes both to microvascular damage and diabetic complications. The aim of this study was to investigate altered leucocyte SA metabolism in diabetic subjects and measure lysosomal sialidase which regulates leucocyte surface sialylation. We examined 26 Type 1 (insulin-dependent) diabetic subjects with retinopathy, 26 Type 1 diabetic subjects without complications, and 38 matched normal control subjects. Sialidase was assayed in freshly prepared sonicates of pure mononuclear leucocytes (MNLs), using the fluorometric substrate 4-methyl-umbelliferyl-N-acetylneuraminic acid. In the subjects with diabetes there was a significant negative correlation between MNL sialidase activity and both HbA1c (rs = 0.37, p = 0.007) and fructosamine (rs = -0.31, p = 0.026). MNL sialidase activity was significantly decreased in diabetic subjects with clinical evidence of complications compared to control subjects. HbA1c was significantly higher (p = 0.036) in diabetic patients with complications compared to those without. The observed decrease in MNL sialidase activity related to diabetic control may be important in the pathogenesis of vascular damage. Diabetes-associated changes in sialylation of functional cell surface glycoconjugates may have important clinical consequences.

Postprandial mesenteric blood flow in humans: relationship to endogenous gastrointestinal hormone secretion and energy content of food.

OBJECTIVE: To examine the relationship between postprandial superior mesenteric arterial blood flow (SMABF) and endogenous gastrointestinal hormone secretion after liquid meals of varying energy content. METHODS: Six healthy volunteers received four isovolumetric meals of differing energy content. SMABF was measured before and for 120 min after feeding using duplex ultrasound; plasma levels of gastrointestinal hormones and noradrenaline were measured. RESULTS: Results are given as means +/- SEM. Postprandial SMABF increased after each meal; the peak mean percentage increment occurred at 28 +/- 8 min, varying from 106 +/- 26% (P < 0.05) with the 800 kj meal to 240 +/- 62% (P < 0.05) with the 4800 kj meal. The rise was sustained and correlated with energy content (r = 0.969; P = 0.031). The subjects' pulse rate increased after each meal and the increment appeared to be related to meal size (r = 0.990; P = 0.008). Blood pressure did not change. The postprandial concentration of N-terminal neurotensin peaked between 30 and 120 min. The mean peak concentration increased from a baseline value of 30.9 +/- 3.5 ng/l by 19.3 ng/l after 800 kj, 31 ng/l after 1600 kj, 38 ng/l after 2400 kj and 64.3 ng/l after the 4800 kj meal (all differences P < 0.05). The rise was sustained and correlated with energy content (r = 0.967; P = 0.033). Plasma noradrenaline concentration also increased significantly after each meal and was related to energy content (r = 0.90; P = 0.036). Plasma insulin concentration showed a similar postprandial response but had no relationship to energy content. Gastrin concentration increased transiently but changes in the concentration of glucagon, vasoactive intestinal polypeptide and neurokinin A were not significant. CONCLUSIONS: The results indicate a relationship between meal energy content, postprandial SMABF and pulse rate increments. Postprandial increases in N-terminal neurotensin and noradrenaline concentration were also related to energy content and may indicate a role for these hormones in the control of SMABF after feeding.

The prevalence of autonomic and peripheral neuropathy in insulin-treated diabetic subjects.

The prevalence of autonomic and peripheral neuropathy was examined in 506 diabetic subjects treated with insulin, mean age 43 years, diabetes duration 15 (range 1-54) years. Autonomic neuropathy was present if two or more (of four) cardiovascular autonomic function tests were abnormal using age-related ranges derived from 310 normal control subjects. Peripheral neuropathy was defined as a vibration threshold > 95th centile for age combined with absent/impaired ankle reflexes. Eighty-four (16.6%) of diabetic subjects had abnormal autonomic function and 119 (23.5%) peripheral neuropathy, concordant in 44/506 (8.7%). Of the diabetic subjects with autonomic neuropathy 40/84 (47.6%) did not have peripheral neuropathy and only 44/119 (37.0%) with peripheral neuropathy had abnormal autonomic function (p < 0.001). The prevalence of both neuropathies increased in relation to diabetes duration (both p < 0.001). Autonomic neuropathy was more common in subjects diagnosed < 20 years of age (18.2%) vs age > 40 years (11.1%) (p < 0.05). In contrast peripheral neuropathy was more common with older age at diagnosis (< 20 years 13.5% vs 36.8% > 40 years, p < 0.001). The age-related prevalence of autonomic neuropathy peaked at age 40-49 years while peripheral neuropathy increased progressively with age (p < 0.001). The prevalence of peripheral exceeded autonomic neuropathy 20 years after diagnosis (40.2% vs 30.7%, p < 0.001).

Prolonged remission of hypercalcaemia due to parathyroid carcinoma with pamidronate.

Parathyroid carcinoma is rare and the associated hypercalcaemia is often resistant to all treatment. A case is described in which prolonged control of hypercalcaemia has been achieved by infrequent infusions of pamidronate despite continuing hypersecretion of parathyroid hormone.

Control of human thyroid autoantibody production in SCID mice.

In order to determine the phenotype of the cells required for thyroid autoantibody production, peripheral blood mononuclear cells (PBMC) from patients with autoimmune thyroid disease (AITD) were transferred to severe combined immunodeficient (SCID) mice. The production of human IgG, thyroglobulin (Tg) antibody and thyroid peroxidase (TPO) antibody in the SCID recipients was monitored for up to 4 months. PBMC from 10 of 13 AITD patients produced substantial IgG (> or = 100 micrograms/ml) and detectable Tg and TPO antibodies in recipient mice. PBMC pretreated to deplete or enrich T cells produced low or undetectable thyroid-specific antibody in SCID mice. Depletion of CD4+ T cells resulted in much lower or undetectable IgG, Tg and TPO antibodies compared with levels seen in recipients of control PBMC. By contrast, depletion of CD8+ T cells from the PBMC had no overall effect on autoantibody production, although with PBMC from some patients CD8+ depletion possibly enhanced both IgG and autoantibody production. In eight of 10 experiments, autoantibody levels reached maximal titres before total IgG levels peaked. It is considered that thyroid autoantibodies are produced from memory B cells activated in SCID mice and that this activation is T cell- and CD4+ T cell-dependent.

Evidence against a putative role for glucagon as a physiological splanchnic vasodilator in man.

1. Previous studies have suggested that glucagon in supraphysiological doses may mediate postprandial and hypoglycaemia-induced splanchnic vasodilatation in man and experimental animals. There are no reported studies investigating the role of glucagon in doses producing circulating concentrations within the physiological range. 2. Two separate studies were performed. In study 1, superior mesenteric artery blood flow was measured by Doppler ultrasound in six normal subjects during either saline or glucagon infusion at 1, 3 and 6 ng min-1kg-1, which resulted in circulating glucagon levels within the physiological range. Mean superior mesenteric artery blood flow fell during the 3 and 6 ng min-1kg-1 glucagon infusions (3 ng min-1kg-1: -31.8%, range -20 to -56% of baseline; 6 ng min-1kg-1: -20.7%, range -8 to -53% of baseline; P < 0.05). 3. In study 2, superior mesenteric artery blood flow was measured during hypoglycaemia induced by an insulin infusion in 12 normal subjects. In six of these subjects the effect of suppression of glucagon release during hypoglycaemia was assessed by pretreatment with the somatostatin analogue octreotide (0.8 microgram/kg subcutaneously) given 30 min before the insulin infusion. 4. The nadir in blood glucose concentration at the hypoglycaemic reaction was similar in both groups and glucose recovery was complete by 60 min after the hypoglycaemic reaction. Plasma catecholamine concentrations rose in both groups after the hypoglycaemic reaction. 5. Superior mesenteric artery blood flow rose at the hypoglycaemic reaction in both groups despite suppression of glucagon release with octreotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Splanchnic blood flow in man: evidence for mediation via a beta-adrenergic mechanism.

1. Superior mesenteric artery blood flow was examined by Doppler ultrasound in six male subjects aged 19-23 years during the infusion of saline (control), 10 and 40 ng of adrenaline min-1kg-1 for 30 min, or propranolol and 10 ng of adrenaline min-1kg-1 for 30 min, on four separate occasions. 2. Adrenaline infusion resulted in significant peak mean (SEM) rises in circulating adrenaline concentrations during the infusion period only [control, 0.20 (0.05) nmol/l; 10 ng of adrenaline min-1kg-1, 1.37 (0.29) nmol/l; 40 ng of adrenaline min-1kg-1, 3.73 (0.40) nmol/l; 10 ng of adrenaline min-1kg-1 and propranolol, 1.48 (0.16) nmol/l, P < 0.001 versus control]. These values are within the physiological range. 3. Superior mesenteric artery blood flow rose in a dose-dependent manner during the adrenaline infusions alone, but not during the infusion of adrenaline and propranolol [mean (95% confidence interval) area under the curve: control, -4.2 (-11 to +2.7)%; 10 ng of adrenaline min-1kg-1, +4 (-1 to 11.9)%; 40 ng of adrenaline min-1kg-1, +34 (+6.5 to +61.5)%; 10 ng of adrenaline min-1kg-1 and propranolol, -8.4 (-23 to +6)%]. 4. Superior mesenteric artery resistance fell during the adrenaline infusions alone and rose during the combined adrenaline and propranolol infusion [mean (SEM) area under the curve: control, 6.4 (2.7)%; 10 ng of adrenaline min-1kg-1, -2.9 (2.5)%; 40 ng of adrenaline min-1kg-1, -15 (1.4)%; 10ng of adrenaline min-1kg-1 and propranolol, 16.9 (10)%]. 5. These data suggest that splanchnic vasodilatation is mediated via a beta-adrenergic mechanism.

Suppression of lipolysis in normal man does not inhibit recovery from insulin-induced hypoglycaemia.

Pharmacological suppression of lipolysis is being increasingly used in the treatment of diabetic hyperlipidaemia. Although theoretical hazard of such treatment is that recovery from hypoglycaemia might be impaired. Seven normal subjects were therefore studied on two occasions, following treatment with a single dose of either acipimox 250 mg or placebo. Hypoglycaemic recovery was unaffected, despite effective suppression of plasma non-esterified fatty acid levels with acipimox. The results suggest that under these conditions activation of lipolysis may not be essential to recovery from hypoglycaemia.