Diagnosis and treatment of anti-insulin antibody-mediated labile glycaemia in insulin-treated diabetes.

AIMS: Anti-insulin antibodies in insulin-treated diabetes can derange glycaemia, but are under-recognised. Detection of significant antibodies is complicated by antigenically distinct insulin analogues. We evaluated a pragmatic biochemical approach to identifying actionable antibodies, and assessed its utility in therapeutic decision making. METHODS: Forty people with insulin-treated diabetes and combinations of insulin resistance, nocturnal/matutinal hypoglycaemia, and unexplained ketoacidosis were studied using broad-specificity insulin immunoassays, polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC) with or without ex vivo insulin preincubation. RESULTS: Twenty-seven people had insulin immunoreactivity (IIR) below 3000 pmol/L that fell less than 50% after PEG precipitation. Insulin binding by antibodies in this group was low and judged insignificant. In 8 people IIR was above 3000 pmol/L and fell by more than 50% after PEG precipitation. GFC demonstrated substantial high molecular weight (HMW) IIR in 7 of these 8. In this group antibodies were judged likely significant. In 2 people immunosuppression was introduced, with a good clinical result in one but only a biochemical response in another. In 6 people adjustment of insulin delivery was subsequently informed by knowledge of underlying antibody. In a final group of 5 participants IIR was below 3000 pmol/L but fell by more than 50% after PEG precipitation. In 4 of these GFC demonstrated low levels of HMW IIR and antibody significance was judged indeterminate. CONCLUSIONS: Anti-insulin antibodies should be considered in insulin-treated diabetes with unexplained glycaemic lability. Combining immunoassays with PEG precipitation can stratify their significance. Antibody depletion may be beneficial, but conservative measures often suffice.

Intermittent exposure of cultured endothelial cells to physiologically relevant fructose concentrations has a profound impact on nitric oxide production and bioenergetics.

Hyperglycaemia is known to induce endothelial dysfunction and changes in metabolic function, which could be implicated in diabetes-induced cardiovascular disease. To date, however, little is known about the impact of physiologically relevant concentrations of fructose on endothelial cells. A novel in vitro model was devised to establish the impact of substitution of a small proportion of glucose with an equal concentration (0.1 mM or 1 mM) of fructose on EA.hy926 endothelial cells during periodic carbohydrate "meals" superimposed on a normoglycaemic (5.5 mM) background. Parallel experiments were conducted using meals consisting of normoglycaemic glucose, intermediate glucose (12.5 mM) or profound hyperglycaemia (25 mM), each delivered for 2 h, with and without substituted fructose over 50 h. Outcome measures included nitrite as a surrogate marker of the mediator of healthy endothelial function, nitric oxide (NO), and a range of bioenergetic parameters using a metabolic analyser. Despite its relatively low proportion of carbohydrate load, intermittent fructose induced a substantial reduction (approximately 90%) in NO generation in cells treated with either concentration of fructose. Cell markers of oxidative stress were not altered by this treatment regimen. However, the cells experienced a marked increase in metabolic activity induced by fructose, irrespective of the glucose concentration delivered simultaneously in the "meals". Indeed, glucose alone failed to induce any metabolic impact in this model. Key metabolic findings were a 2-fold increase in basal oxygen consumption rate and a similar change in extracellular acidification rate-a marker of glycolysis. Non-metabolic oxygen consumption also increased substantially in cells exposed to fructose. There was no difference between results with 0.1 mM fructose and those with 1 mM fructose. Low, physiologically relevant concentrations of fructose, delivered in a pattern that mimics mealtime consumption, had a profound impact on endothelial function and bioenergetics in an in vitro cell model. The results suggest that endothelial cells are exquisitely sensitive to circulating fructose; the potential ensuing dysfunction could have major implications for development of atherosclerotic disease associated with high fructose consumption.

The impact of glucose exposure on bioenergetics and function in a cultured endothelial cell model and the implications for cardiovascular health in diabetes.

Cardiovascular disease is the primary driver of morbidity and mortality associated with diabetes. Hyperglycaemia is implicated in driving endothelial dysfunction that might underpin the link between diabetes and cardiovascular disease. This study was designed to determine the impact of chronic preconditioning of cells to hyperglycaemia and transient switching of cultured endothelial cells between hyper- and normo-glycaemic conditions on bioenergetic and functional parameters. Immortalised EA.hy926 endothelial cells were cultured through multiple passages under normoglycaemic (5.5 mM) or hyperglycaemic (25 mM) conditions. Cells were subsequently subjected (48 h) to continued normo- or hyperglycaemic exposure, or were switched to the alternative glycaemic condition, or to an intermediate glucose concentration (12.5 mM) and metabolic activity, together with key markers of function were measured. Cells habituated to hyperglycaemia were energetically quiescent. Functional activity, characterised by the measurement of nitric oxide, endothelin-1, tissue plasminogen activator and plasminogen activator inhibitor-1, was depressed by exposure to high glucose, with the reduction in nitric oxide production being the most notable. Function was more responsive to acute changes in extracellular glucose than were bioenergetic changes. We conclude that glucose is a key determinant of endothelial function. The study highlights the importance of chronic glucose exposure on cell phenotype and emphasises the need to pay close attention to glucose preconditioning in interpreting results under culture conditions.

Acute physiological effects of glucocorticoids on fuel metabolism in humans are permissive but not direct.

BACKGROUND AND AIMS: The effects of glucocorticoids on fuel metabolism are complex. Acute glucocorticoid excess promotes lipolysis but chronic glucocorticoid excess causes visceral fat accumulation. We hypothesized that interactions between cortisol and insulin and adrenaline account for these conflicting results. We tested the effect of cortisol on lipolysis and glucose production with and without insulin and adrenaline in humans both in vivo and in vitro. MATERIALS AND METHODS: A total of 20 healthy men were randomized to low and high insulin groups (both n = 10). Subjects attended on 3 occasions and received low (c. 150 nM), medium (c. 400 nM) or high (c. 1400 nM) cortisol infusion in a randomized crossover design. Deuterated glucose and glycerol were infused intravenously along with a pancreatic clamp (somatostatin with replacement of glucagon, insulin and growth hormone) and adrenaline. Subcutaneous adipose tissue was obtained for analysis. In parallel, the effect of cortisol on lipolysis was tested in paired primary cultures of human subcutaneous and visceral adipocytes. RESULTS: In vivo, high cortisol increased lipolysis only in the presence of high insulin and/or adrenaline but did not alter glucose kinetics. High cortisol increased adipose mRNA levels of ATGL, HSL and CGI-58 and suppressed G0S2. In vitro, high cortisol increased lipolysis in the presence of insulin in subcutaneous, but not visceral, adipocytes. CONCLUSIONS: The acute lipolytic effects of cortisol require supraphysiological concentrations, are dependent on insulin and adrenaline and are observed only in subcutaneous adipose tissue. The resistance of visceral adipose tissue to cortisol's lipolytic effects may contribute to the central fat accumulation observed with chronic glucocorticoid excess.

Asymptomatic and mild primary hyperparathyroidism.

Primary hyperparathyroidism is common, with epidemiological studies suggesting it may effect up to 1% of the population, and up to 3% of post-menopausal females. Many cases are diagnosed incidentally on routine blood sampling, with the majority of patients being seemingly asymptomatic at diagnosis, and often having mild hypercalcaemia of <2.85mmol/L. Individuals with this so-called mild or asymptomatic primary hyperparathyroidism may however have undiagnosed complications, along with subtle neuropsychological symptoms, and possibly increased cardiovascular risk. Revised international guidelines suggest a more proactive approach to screening for vertebral fractures and renal stones, although the thresholds for recommending definitive surgical management with parathyroidectomy remain similar. The natural history of mild primary hyperparathyroidism will be reviewed, with particular reference to the Parathyroid Epidemiology and Audit Research Study (PEARS), which has used population level data from Tayside, Scotland (UK), along with recent data describing an association with cardiovascular disease. Results of intervention studies, including randomised controlled trials, will also be discussed.

5α-Reductase type 1 deficiency or inhibition predisposes to insulin resistance, hepatic steatosis, and liver fibrosis in rodents.

5α-Reductase type 1 (5αR1) catalyses A-ring reduction of androgens and glucocorticoids in liver, potentially influencing hepatic manifestations of the metabolic syndrome. Male mice, homozygous for a disrupted 5αR1 allele (5αR1 knockout [KO] mice), were studied after metabolic (high-fat diet) and fibrotic (carbon tetrachloride [CCl4]) challenge. The effect of the 5α-reductase inhibitor finasteride on metabolism was investigated in male obese Zucker rats. While eating a high-fat diet, male 5αR1-KO mice demonstrated greater mean weight gain (21.6 ± 1.4 vs 16.2 ± 2.4 g), hyperinsulinemia (insulin area under the curve during glucose tolerance test 609 ± 103 vs. 313 ± 66 ng ⋅ mL(-1) ⋅ min), and hepatic steatosis (liver triglycerides 136.1 ± 17.0 vs. 89.3 ± 12.1 µmol ⋅ g(-1)). mRNA transcript profiles in liver were consistent with decreased fatty acid ß-oxidation and increased triglyceride storage. 5αR1-KO male mice were more susceptible to fibrosis after CCl4 administration (37% increase in collagen staining). The nonselective 5α-reductase inhibitor finasteride induced hyperinsulinemia and hepatic steatosis (10.6 ± 1.2 vs. 7.0 ± 1.0 µmol ⋅ g(-1)) in obese male Zucker rats, both intact and castrated. 5αR1 deficiency induces insulin resistance and hepatic steatosis, consistent with the intrahepatic accumulation of glucocorticoids, and predisposes to hepatic fibrosis. Hepatic steatosis is independent of androgens in rats. Variations in 5αR1 activity in obesity and with nonselective 5α-reductase inhibition in men with prostate disease may have important consequences for the onset and progression of metabolic liver disease.

5α-reductase type 1 modulates insulin sensitivity in men.

CONTEXT: 5α-Reductase (5αR) types 1 and 2 catalyze the A-ring reduction of steroids, including androgens and glucocorticoids. 5α-R inhibitors lower dihydrotestosterone in benign prostatic hyperplasia; finasteride inhibits 5αR2, and dutasteride inhibits both 5αR2 and 5αR1. In rodents, loss of 5αR1 promotes fatty liver. OBJECTIVE: Our objective was to test the hypothesis that inhibition of 5αR1 causes metabolic dysfunction in humans. DESIGN, SETTING, AND PARTICIPANTS: This double-blind randomized controlled parallel group study at a clinical research facility included 46 men (20-85 years) studied before and after intervention. INTERVENTION: Oral dutasteride (0.5 mg daily; n = 16), finasteride (5 mg daily; n = 16), or control (tamsulosin; 0.4 mg daily; n = 14) was administered for 3 months. MAIN OUTCOME MEASURE: Glucose disposal was measured during a stepwise hyperinsulinemic-euglycemic clamp. Data are mean (SEM). RESULTS: Dutasteride and finasteride had similar effects on steroid profiles, with reduced urinary androgen and glucocorticoid metabolites and reduced circulating DHT but no change in plasma or salivary cortisol. Dutasteride, but not finasteride, reduced stimulation of glucose disposal by high-dose insulin (dutasteride by -5.7 [3.2] µmol/kg fat-free mass/min, versus finasteride +7.2 [3.0], and tamsulosin +7.0 [2.0]). Dutasteride also reduced suppression of nonesterified fatty acids by insulin and increased body fat (by 1.6% [0.6%]). Glucose production and glycerol turnover were unchanged. Consistent with metabolic effects of dutasteride being mediated in peripheral tissues, mRNA for 5αR1 but not 5αR2 was detected in human adipose tissue. CONCLUSION: Dual inhibition of 5αRs, but not inhibition of 5αR2 alone, modulates insulin sensitivity in human peripheral tissues rather than liver. This may have important implications for patients prescribed dutasteride for prostatic disease.

Subclinical and asymptomatic parathyroid disease: implications of emerging data.

Primary hyperparathyroidism, a disorder in which there is a tendency for hypercalcaemia caused by autonomous overproduction of parathyroid hormone, is common, especially in postmenopausal women. Although parathyroidectomy is indicated for symptomatic patients, most individuals with the disorder are asymptomatic and without classic complications, such as renal stones and osteoporosis, at diagnosis. Consensus guidelines suggest which individuals might be suitable for medical follow-up rather than parathyroidectomy, but there are no long-term randomised controlled trials to support the safety of medical surveillance, and some patients progress with time. Data from observational studies suggest that cardiovascular morbidity and mortality are increased in patients with primary hyperparathyroidism, and might be predicted by parathyroid hormone concentrations, even in individuals with asymptomatic primary hyperparathyroidism. Whether parathyroidectomy improves cardiovascular outcomes in patients with asymptomatic primary hyperparathyroidism is unproven, but data suggest that surgery decreases fracture risk and might improve neuropsychological symptoms. Studies also show that patients with normocalcaemic (subclinical) hyperparathyroidism and hypoparathyroidism have a low risk of progression to overt disease, but their long-term risks are not defined. In this Review, we explore the increasing range of asymptomatic parathyroid disorders, focusing on current evidence about their natural history and potential complications, with a particular emphasis on primary hyperparathyroidism.

Should 'mild primary hyperparathyroidism' be reclassified as 'insidious': is it time to reconsider?

Primary hyperparathyroidism (PHPT) is a common incidental finding on routine biochemical testing, affecting around 1% of the population. The majority of individuals will be asymptomatic at diagnosis, with no evidence of end organ damage, and unless individuals aged <50 years at diagnosis, they are often considered to have 'mild' PHPT, as they do not meet published criteria for parathyroidectomy (PTX). However, there is increasing evidence that 'mild' PHPT is associated with adverse health outcomes. Long-term observational studies describing the natural history of 'mild' PHPT suggest that even though biochemistry may be relatively stable in the majority, bone mineral density (BMD) does decline after approximately 10 years of observation, whereas significant improvements in BMD are seen following PTX. Recent large European record linkage studies of 'mild PHPT' demonstrate significantly increased all-cause and cardiovascular mortality, similar to rates published for patients with PHPT who meet the NIH surgical criteria. 'Mild' PHPT was also associated with increased admissions for nonfatal cardiovascular disease, renal failure, renal stones, fractures, hypertension, psychiatric disease, cancer and diabetes, suggesting that 'insidious' PHPT may be a more appropriate description, or at least that the term 'mild' should be abandoned. Randomized controlled trials (RCTs) have begun to explore the benefits of PTX in this condition, demonstrating improvements in BMD and some psychiatric outcomes at approximately 2 years of follow-up. However, larger, adequately powered, long-term, RCTs will be required to determine whether PTX improves potential long-term morbidity and mortality in patients with PHPT who do not meet standard surgical criteria.

Metabolic pathways promoting intrahepatic fatty acid accumulation in methionine and choline deficiency: implications for the pathogenesis of steatohepatitis.

The pathological mechanisms that distinguish simple steatosis from steatohepatitis (or NASH, with consequent risk of cirrhosis and hepatocellular cancer) remain incompletely defined. Whereas both a methionine- and choline-deficient diet (MCDD) and a choline-deficient diet (CDD) lead to hepatic triglyceride accumulation, MCDD alone is associated with hepatic insulin resistance and inflammation (steatohepatitis). We used metabolic tracer techniques, including stable isotope ([¹³C4]palmitate) dilution and mass isotopomer distribution analysis (MIDA) of [¹³C2]acetate, to define differences in intrahepatic fatty acid metabolism that could explain the contrasting effect of MCDD and CDD on NASH in C57Bl6 mice. Compared with control-supplemented (CS) diet, liver triglyceride pool sizes were similarly elevated in CDD and MCDD groups (24.37 ± 2.4, 45.94 ± 3.9, and 43.30 ± 3.5 µmol/liver for CS, CDD, and MCDD, respectively), but intrahepatic neutrophil infiltration and plasma alanine aminotransferase (31 ± 3, 48 ± 4, 231 ± 79 U/l, P < 0.05) were elevated only in MCDD mice. However, despite loss of peripheral fat in MCDD mice, neither the rate of appearance of palmitate (27.2 ± 3.5, 26.3 ± 2.3, and 28.3 ± 3.5 µmol·kg⁻¹·min⁻¹) nor the contribution of circulating fatty acids to the liver triglyceride pool differed between groups. Unlike CDD, MCDD had a defect in hepatic triglyceride export that was confirmed using intravenous tyloxapol (142 ± 21, 122 ± 15, and 80 ± 7 mg·kg⁻¹·h⁻¹, P < 0.05). Moreover, hepatic de novo lipogenesis was significantly elevated in the MCDD group only (1.4 ± 0.3, 2.3 ± 0.4, and 3.4 ± 0.4 µmol/day, P < 0.01). These findings suggest that important alterations in hepatic fatty acid metabolism may promote the development of steatohepatitis. Similar mechanisms may predispose to hepatocyte damage in human NASH.

One-hour plasma glucose identifies insulin resistance and beta-cell dysfunction in individuals with normal glucose tolerance: cross-sectional data from the Relationship between Insulin Sensitivity and Cardiovascular Risk (RISC) study.

OBJECTIVE: Some individuals with normal glucose tolerance (NGT) exhibit a 1-h excursion of plasma glucose during oral glucose tolerance testing as high as that of individuals with impaired glucose tolerance (IGT). The aim of this study was to characterize their metabolic phenotype. RESEARCH DESIGN AND METHODS: A total of 1,205 healthy volunteers (aged 29-61 years) underwent assessment of 1) oral glucose tolerance and 2) insulin sensitivity (standardized euglycemic-hyperinsulinemic clamp), as part of the Relationship between Insulin Sensitivity and Cardiovascular Risk (RISC) study. RESULTS: One-hour plasma glucose correlated better than 2-h plasma glucose with total insulin secretion (r = 0.43), beta-cell glucose sensitivity (r = -0.46), and beta-cell rate sensitivity (r = -0.18). Receiver operating characteristic analysis identified 8.95 mmol/l as the best cutoff value for prediction of IGT from 1-h plasma glucose (sensitivity 77% and specificity 80%). Participants with NGT with 1-h plasma glucose >8.95 mmol/l had larger waist circumference, higher BMI, lower insulin sensitivity, higher fasting glucose, and higher insulin secretion than their counterparts with 1-h plasma glucose

Bile acids modulate glucocorticoid metabolism and the hypothalamic-pituitary-adrenal axis in obstructive jaundice.

BACKGROUND & AIMS: Suppression of the hypothalamic-pituitary-adrenal axis occurs in cirrhosis and cholestasis and is associated with increased concentrations of bile acids. We investigated whether this was mediated through bile acids acting to impair steroid clearance by inhibiting glucocorticoid metabolism by 5beta-reductase. METHODS: The effect of bile acids on glucocorticoid metabolism was studied in vitro in hepatic subcellular fractions and hepatoma cells, allowing quantitation of the kinetics and transcript abundance of 5beta-reductase. Metabolism was subsequently examined in vivo in rats following dietary manipulation or bile duct ligation. Finally, glucocorticoid metabolism was assessed in humans with obstructive jaundice. RESULTS: In rat hepatic cytosol, chenodeoxycholic acid competitively inhibited 5beta-reductase (K(i) 9.19+/-0.40 microM) and reduced its transcript abundance (in H4iiE cells) and promoter activity (reporter system, HepG2 cells). In Wistar rats, dietary chenodeoxycholic acid (1% w/w chow) inhibited hepatic 5beta-reductase activity, reduced urinary excretion of 3alpha,5beta-tetrahydrocorticosterone and reduced adrenal weight. Conversely, a fat-free diet suppressed bile acid levels and increased hepatic 5beta-reductase activity, supplementation of the fat-free diet with CDCA reduced 5beta-reductase activity, and urinary 3alpha,5beta-reduced corticosterone. Cholestasis in rats suppressed hepatic 5beta-reductase activity and transcript abundance. In eight women with obstructive jaundice, relative urinary excretion of 3alpha,5beta-tetrahydrocortisol was significantly lower than in healthy controls. CONCLUSION: These data suggest a novel role for bile acids in inhibiting hepatic glucocorticoid clearance, of sufficient magnitude to suppress hypothalamic-pituitary-adrenal axis activity. Elevated hepatic bile acids may account for adrenal insufficiency in liver disease.

Glucocorticoids and fatty acid metabolism in humans: fuelling fat redistribution in the metabolic syndrome.

Glucocorticoid hormones constitute an integral component of the response to stress, and many of the manifestations of glucocorticoid excess (Cushing's syndrome) are predictable on the basis of their acute effects to raise blood pressure, induce insulin resistance, increase protein catabolism and elevate plasma glucose. However, it appears to be a paradox that the acute lipolytic effect of glucocorticoids is not manifest in long-term weight loss in humans. The effects of glucocorticoids on glucose metabolism are well characterised, involving impaired peripheral glucose uptake and hepatic insulin resistance, and there is mounting evidence that subtle abnormalities in glucocorticoid concentrations in the plasma and/or in tissue sensitivity to glucocorticoids are important in metabolic syndrome. The effects of glucocorticoids on fatty acid metabolism are less well understood than their influence on glucose metabolism. In this article, we review the literature describing the effects of glucocorticoids on fatty acid metabolism, with particular reference to in vivo human studies. We consider the implications for contrasting acute versus chronic effects of glucocorticoids on fat accumulation, effects in different adipose depots and the potential role of glucocorticoid signalling in the pathogenesis and therapy of metabolic syndrome.

Thiazolidinediones in patients with diabetes mellitus and heart failure : implications of emerging data.

Individuals with diabetes mellitus have an increased risk of developing heart failure, usually as a consequence of coronary artery disease, although a specific diabetic cardiomyopathy, secondary to a microangiopathy, may also exist. The thiazolidinediones, a relatively new class of insulin-sensitizing agents used in the management of type 2 diabetes mellitus, have a number of complex metabolic actions on surrogate markers of atherogenesis, supported by the results of the recently published PROACTIVE (PROspective pioglitAzone Clinical Trial In macroVascular Events) trial. Unfortunately, the use of thiazolidinediones in individuals with diabetes mellitus and heart failure is limited because of a propensity to cause fluid retention. The underlying mechanisms of fluid retention have yet to be fully elucidated, but appear to be a dose-related class effect, exacerbated by combination therapy with insulin, and in some cases may be localized to peripheral edema. In parallel, echocardiographic studies show no significant effect of thiazolidinediones on cardiac structure or function. The design of epidemiologic studies describing an increased risk of developing heart failure in individuals with type 2 diabetes mellitus prescribed thiazolidinediones has been questioned, and a study of 'new users' of antihyperglycemic treatments found no increased risk of hospitalization for heart failure with thiazolidinedione therapy. There is also increasing evidence for the potential benefits of insulin sensitization in patients with diabetes mellitus and known heart failure, and a large observational study of over 16 000 patients with a principal discharge diagnosis of heart failure found a reduced mortality (hazard ratio [HR] 0.87; 95% CI 0.80, 0.94) in those prescribed thiazolidinediones. This benefit was offset by an increased risk of readmission with heart failure (HR 1.06; 95% CI 1.00, 1.09). Despite an increase in fluid-related events, recent studies suggest that individuals with type 2 diabetes mellitus and heart failure (New York Heart Association grade I/II) can be treated with thiazolidinediones with appropriate monitoring and adjustment of heart failure therapies. These findings would suggest the need for large-scale, prospective trials to investigate the safety and potential benefits of thiazolidinedione use in patients with diabetes mellitus and heart failure.

A rare and life threatening complication of prosthetic valve endocarditis.

Up to 70% of cases of Guillain-Barré syndrome (GBS) follow a preceding infection and a number of infectious agents have been described. We present a previously unreported association of Streptococcus viridans infective endocarditis affecting a prosthetic aortic valve and Guillain-Barré syndrome. This case highlights that this potentially life-threatening diagnosis should be considered in any patient presenting with symptoms of peripheral nervous system dysfunction following an infective illness.