John A. Charlesworth

The postprandial response of adiponectin to a high-fat meal in normal and insulin-resistant subjects

OBJECTIVE: Adiponectin is an adipose-specific protein with short-term effects in vivo on glucose and fatty acid levels. We studied the plasma concentration and the proteolytic activation status of adiponectin following the consumption of a high-fat, low-carbohydrate meal.DESIGN: Analysis of adiponectin concentration and polypeptide structure after consumption of a fat meal.SUBJECTS: Normal subjects (n=24) and first-degree relatives of patients with type II diabetes (n=20).MEASUREMENTS: All subjects had a normal fasting plasma glucose and glucose tolerance. Blood was collected for the determination of plasma insulin, adiponectin, triglyceride, and free fatty acids. Body composition was assessed with dual-energy X-ray absorptiometry and whole-body insulin sensitivity with a euglycaemic, hyperinsulinaemic clamp. Postprandial response over 6 h was determined for plasma adiponectin, glucose, insulin, triglyceride, and free fatty acids. Adiponectin was measured by commercial RIA and its polypeptide structure examined by Western blotting.RESULTS: The relatives were more insulin resistant and had increased adiposity compared with control subjects. There was no significant difference in postprandial response in fatty acids, triglyceride, or insulin between the groups. Postprandial levels of adiponectin measured by radioimmunoassay were not significantly different from fasting levels, and no breakdown products of adiponectin were detectable in postprandial samples by Western blotting.CONCLUSIONS: Levels of circulating adiponectin do not alter in response to a fat meal, despite evidence in mice that acute changes in adiponectin significantly affect postprandial fatty acid flux. Moreover, a fat meal challenge did not lead to significant activation of adiponectin by proteolytic conversion.

The inhibitory effect of rosmarinic acid on complement involves the C5 convertase

Rosmarinic acid (RA), a naturally occurring extract from Melissa officinalis, inhibits several complement-dependent inflammatory processes and may have potential as a therapeutic agent for the control of complement activation in disease. Rosmarinic acid has been reported to have effects on both the classical pathway C3-convertase and on the cobra venom factor-induced, alternative pathway convertase. In order to define the mechanism of inhibition, the effect of RA on classical and alternative pathway lysis, C1q binding, the classical pathway convertase, the alternative pathway convertase, membrane attack pathway lysis and the generation of fragments of C3 and C5 during activation, was tested in vitro. The results showed that RA inhibited lysis by the classical pathway more than by the alternative pathway. This effect was dose-dependent with maximum inhibition of classical pathway lysis observed at 2.6 mmoles of RA. There was little effect on C1q binding or on the classical and alternative pathway convertases. However, there was highly significant inhibition of lysis of pre-formed EA43b cells by dilutions of human or rabbit serum in the presence of RA (1 mM); this was accompanied by inhibition of C5a generation. We conclude that the inhibitory effect of RA involves the C5 convertase. Such inhibition could be advantageous to the host in disorders where the terminal attack sequence plays a role in pathogenesis.

Vitronectin‐mediated inhibition of complement: evidence for different binding sites for C5b‐7 and C9

In the activated complement system, vitronectin (complement S‐protein) occupies the metaslable membrane binding site of the nascent precursor complex C5b‐7, so that the newly formed SC5b‐7 is unable to insert into cell membranes. Some evidence also indicates that vilronectin limits on‐going membrane‐associated pore formation by inhibiting C9 polymerization. It has been assumed that these two stages of terminal complement complex (TCC) inhibition take place through charge interactions between the heparin‐binding region of vitronectin and homologous cysteine‐rich sequences of the late complement proteins C6, C7, C8 and C9. We examined SC5b‐7 formation and inhibition of C9 binding in the TCC using separate haemolytic assays. The mode of action of vitronectin in these assays was compared with two 15mer peptides which span residues 348–379 of the heparin‐binding region, and a heparin‐attinity polypeptide. protamine sulphate. The results showed that vitronectin acts predominantly through SC5b‐7 production with a lesser effect on the inhibition of C9 lytic pore formation. In contrast, protamine sulphate did not prevent C5b‐7 membrane attachment, but was a potent inhibitor of C9‐mediated lysis. The peptides did not inhibit C5b‐7 membrane insertion and only one affected C9 binding. These data suggest that the two stages of TCC inhibition involve separate binding sites on the vitronectin molecule. The site for association with nascent C5b‐7 is unknown, whereas inhibition of C9 binding and pore formation lakes place through the heparin‐binding region.

Detection and quantification of the control proteins of the alternative pathway of complement in 3T3-L1 adipocytes

The complement peptide C3a desarg is identical to acylation‐stimulating protein (ASP), a human plasma protein that potently stimulates adipocyte triacylglycerol synthesis and glucose transport. Both human and murine adipocytes express mRNA and/or protein for the complement components C3 and factors B and D (adipsin) required to generate ASP. However, the regulatory mechanisms controlling this process are unknown. We have established a semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR) technique to demonstrate the presence in mouse 3T3‐L1 adipocytes of mRNA for all components of the alternative pathway, including the control proteins factors I and H, CR1 and properdin. On differentiation, mRNA for C3 (fivefold) and factor D (> 50‐fold) increased, whereas stimulation with tumour necrosis factor (TNF)‐α and interleukin (IL) 1β led to eightfold increases in factor B mRNA. Metabolic labelling followed by immunoprecipitation showed that factor B protein is normally present in small quantities, and is greatly increased by cytokine stimulation. The larger quantities of C3 and H proteins present were little affected, whereas levels of C3a increased on cytokine stimulation. These results suggest that the rate‐limiting step in the cytokine‐induced production of ASP in adipocytes is factor B synthesis.

Adiponectin binds C1q and activates the classical pathway of complement.

The adipose-specific protein adiponectin binds to a number of target molecules, including damaged endothelium and the surface of apoptotic cells. However, the significance of this binding remains unclear. This study demonstrates the binding of purified C1q to recombinant adiponectin under physiological conditions, and the dependence of this upon Ca(++) and Mg(++). Binding was enhanced by metaperiodate-mediated destruction of glucosylgalactosyl sugars on adiponectin. Adiponectin was bound by the globular domain of the A chain of collagenase-digested C1q, and C1q binding induced deposition of C4 and C3 through activation of the classical complement pathway. After Western blotting, affinity-purified adiponectin from human serum bound C1q, whereas adiponectin in whole serum did not, unless pre-treated with metaperiodate. These results suggest adiponectin is member of the pattern-recognition family of defence collagens, able to bind target molecules and activate complement. It may therefore play an important role in innate immunity and autoimmune phenomena.

THE SKIN AND IMMUNOSUPPRESSION

Of 135 kidney allograft recipients twenty one (15.5%) developed a total of 84 malignant skin tumours from 4 to 115 months after transplantation. Seven of these patients also developed a total of 12 keratoacanthomas. Skin lesions due to viral, fungal and opportunistic organisms were prevalent but serious pyogenic skin infections were not increased.

The influence of oral lipid loads on acylation stimulating protein (ASP) in healthy volunteers.

OBJECTIVES: To examine the hypothesis that a sustained rise in plasma acylation stimulating protein (ASP, C3a desarg) accompanies the elevation in triacylglycerol that follows the ingestion of an oral fat load.DESIGN: Following an overnight fast, blood samples were obtained from healthy volunteers while fasting and 15 min, 1, 2, 4, 6 and 8 h following ingestion of: (i) a liquid meal, rich in dairy fat (eight subjects) and (ii) a semi-liquid meal, with higher total fat content and rich in polyunsaturated fat (six subjects).SUBJECTS AND METHODS: Four male and four female volunteers (age range: 22–51 y; body mass index (BMI): 17.9–26.9 kg/m2) received the first meal. Six subjects (age range: 32–60 y; BMI: 18.0–28.4 kg/m2), including three from the first study, received the second meal using the same protocol. ASP and C5a were measured by radioimmunoassay (RIA) and the complement proteins C3, factor B and C5 by radial immunodiffusion or nephelometry. Tumour necrosis factor (TNF)-α was measured by enhanced ELISA, and plasma cholesterol and triacylglycerol by an automated enzymatic method. The presence of chylomicrons was assessed in post-prandial plasma samples taken after the second meal.RESULTS: There was no significant change in mean ASP concentration in either group at any time point, following ingestion of either meal. However, there was a significant positive linear trend in ASP following the second fat challenge (ANOVA; P<0.05). There was also no change in complement proteins, plasma cholesterol or TNF-α. Plasma triacylglycerol rose significantly after the first and second meals (P<0.05 and P<0.001 at 2 h post-prandially); the mean maximum rise above the fasting level was 58±41% and 89±38% respectively (mean±s.d.). Chylomicrons were detected in samples taken from each subject after the second meal. Analysis of individual ASP data showed a sustained rise in one subject after the first meal and two subjects after the second meal. Substantial variation in ASP concentration was observed in samples taken in the first 2 h post-prandially.CONCLUSION: There was no significant change in ASP nor other complement proteins for either group of subjects following ingestion of the lipid loads. Individual data showed substantial variation in post-prandial ASP, but multiple plasma sampling did not define the basis for this variation.

Sensory nerve excitability and neuropathy in end stage kidney disease

Background: Peripheral neuropathy is present in 65% of patients with end stage kidney disease (ESKD) starting dialysis. Studies of membrane potential and axonal ion channel function may help explain the pathophysiology. Objectives: To follow changes in median sensory axon excitability in patients with ESKD treated with haemodialysis, and correlate them with clinical rating scales and serum levels of potential neurotoxins. Methods: Sensory nerve action potentials were recorded from the second digit following stimulation of the median nerve in 12 ESKD patients. Stimulus–response behaviour using two stimulus durations, threshold electrotonus to 100 ms polarising currents, a current–threshold relation, and recovery of excitability following supramaximal stimulation were recorded before, during, and after haemodialysis. Serum concentrations of potential neurotoxins were measured. Results: Before dialysis, there were changes in nerve excitability consistent with axonal depolarisation: refractoriness was increased; superexcitability and depolarising threshold electrotonus were reduced. Following dialysis there were improvements in all indices, with correlations between excitability abnormalities and serum potassium measurements. Neuropathic symptoms correlated with excitability changes. Conclusions: Nerves are depolarised before haemodialysis in ESKD patients. The correlation of excitability abnormalities with potassium indicates that the achievement of normokalaemia should be a priority in treating such patients.

Glycosylation of human adiponectin affects its conformation and stability

The collagenous region of adiponectin is glycosylated in vitro with glucosylgalactosyl moieties on four conserved lysines. We investigated the glycosylation of human adiponectin in vivo. Sugar vicinyl hydroxides on adiponectin were oxidized with 10 or 1 mM metaperiodate, and the result analyzed by two-dimensional electrophoresis and immunoblotting. Only 10 mM metaperiodate caused significant changes in electrophoretic mobility and an altered susceptibility to proteinase K digestion. Such treatment also increased the susceptibility of hexamers and high molecular weight (HMW) isoforms to dissociation by SDS. By contrast, untreated low molecular weight (LMW) isoforms were readily dissociated by low concentrations of SDS. Reduced HMW isoforms were able to partially reassemble following the removal of dithiothreitol, and this process was unaffected by metaperiodate. The presence of sialic acid was detected by Maackia amurensis Lectin II blotting, and by oxidation with 1 mM metaperiodate, followed by detection with Emerald Green 300 fluorescent dye. Quantitation of sugars on affinity-purified adiponectin from nine human plasmas showed that dimers of HMW isoforms contained a 1.3-fold greater amount of total sugar than LMW isoforms. However, both contained similar amounts of sialic acid. We conclude that glucosylgalactosyl residues contribute to the conformation of HMW human plasma adiponectin. In addition, the HMW isoform contains greater amounts of glucosylgalactosyl residues than the LMW isoform, and these sugars are important in determining its stability in vivo.

Response of the alternative complement pathway to an oral fat load in first-degree relatives of subjects with type II diabetes

OBJECTIVE:To investigate levels of components of the alternative pathway of complement, and of two activation products, ASP and Bb, in persons ranging in insulin resistance both fasting and following the consumption of a high-fat, low-carbohydrate meal.SUBJECTS:Healthy controls (n=17) and normoglycaemic first-degree relatives of patients with type II diabetes (n=15).MEASUREMENTS:All subjects had normal glucose tolerance. Blood was collected for the measurement of plasma glucose, insulin, triglycerides and free fatty acids. Body composition was assessed with dual energy X-ray absorptiometry (DEXA) and whole-body insulin sensitivity with a euglycaemic, hyperinsulinaemic clamp. Basal and postprandial values over 6 h were determined for plasma C3, B, D, Bb and ASP. Basal levels of C1q, C4 and CRP were also determined.RESULTS:Controls did not differ significantly from the relatives of patients with type II diabetes for any metabolic parameter except in their degree of insulin resistance and central fat (kg). Across all subjects, basal levels of C3, but no other complement protein, were correlated with insulin resistance. Native complement proteins, but not ASP or Bb, were correlated with body mass index and the amount (kg) of central fat. Basal levels of C3 and factor B were significantly higher in the relatives group, whereas factor D and the classical pathway proteins C1q and C4 did not differ between the two groups. Postprandially, levels of factor D were significantly reduced in both groups. ASP levels also fell postprandially, the decline achieving significance in the relatives group.CONCLUSIONS:Elevated levels of C3 and factor B in the diabetic relatives group may have resulted from increased synthesis by adipose tissue. There was no evidence of alternative pathway activation in response to a fat meal in terms of ASP or Bb production, or significant consumption of C3 and factor B. These data do not support an essential requirement of the hypothesis that ASP is produced in response to the intake of fat.