Plasma interleukin-6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes.

Type 2 diabetes is associated with increased circulating concentrations of markers of the acute-phase response and interleukin-6 (IL-6). An augmented acute-phase response may be a mechanism which explains many of the clinical and biochemical features of type 2 diabetes and its complications. We sought to confirm that circulating concentrations of the cytokine acute-phase mediators IL-6 and tumour necrosis factor alpha [TNFalpha] are elevated in type 2 diabetes, and investigated blood as a source of cytokines in type 2 diabetes. Blood samples from 20 type 2 diabetic and 17 age-matched healthy subjects were incubated in vitro for 24 hr with and without lipopolysaccharide (LPS) stimulation and secreted cytokines measured. Plasma IL-6 and TNFalpha were significantly increased in type 2 diabetes compared to normal subjects. However, basal production of IL-6 and TNFalpha in cultured diabetic blood was markedly depressed in comparison with non-diabetic samples. IL-6 and TNFalpha production was increased in blood in response to LPS, reaching similar levels in diabetic and non-diabetic subjects, though IL-6 was slightly but significantly higher in controls. We conclude that circulating levels of IL-6 and TNFalpha are increased in type 2 diabetes but there is downregulation of basal cytokine production in blood cells in type 2 diabetes. Blood has the capacity to produce cytokines in diabetes which contribute to the augmented acute-phase response, but the main source of the increased plasma IL-6 and TNFalpha concentrations may be from non-circulating cells.

The innate immune response and type 2 diabetes: evidence that leptin is associated with a stress-related (acute-phase) reaction.

OBJECTIVE: Leptin is produced by adipose tissue and controls food intake and body weight. Although blood levels of leptin reflect energy stores, cytokines also stimulate leptin production from fat. Because we have proposed that type 2 diabetes mellitus is associated with a cytokine-mediated acute-phase or stress response, part of the innate immune system, we sought evidence that leptin is increased in type 2 diabetes partly as a stress response, independently of obesity and sex. DESIGN: We selected two groups of type 2 diabetic patients with either a low acute-phase response (< 2.30 mmol/l serum concentration of the acute-phase marker sialic acid) or high response (> 2.30 mmol/l sialic acid), but pair-matched for body mass index (BMI) and sex. PATIENTS: Twenty type 2 diabetic subjects (11 male, 9 female) in each group, whose body mass index (BMI) and age were comparable (mean +/- SD: 28.8 +/- 3.8 vs. 28.9 +/- 3.8 kg/m2, and 60.7 +/- 8.9 vs. 61.9 +/- 12.3 years, low vs. high acute-phase responders, respectively). The glycaemic control was also similar in each group (glycated haemoglobin: 9.1 +/- 2.2 vs. 8.9 +/- 1.9%). MEASUREMENTS: Serum concentrations of sialic acid, leptin, interleukin-6 (IL-6) (the major cytokine mediator of the acute-phase response) and cortisol were assayed in fasting venous blood samples from patients and the results compared. RESULTS: Serum leptin concentration was increased in the high compared to the low acute-phase group (median 13.2 (range 3.6-55) vs. 8.1 (2.0-22.5) microg/l, P = 0.004). IL-6 and cortisol concentrations were also higher in the high-stress group (1.9 (1.0-6.4) vs. 1.4 (0.4-7.5) ng/l, P = 0.02; and 409 (180-875) vs. 290 (157-705) nmol/l, P = 0.02, respectively). Leptin was strongly correlated with BMI (r = 0.61, P < 0.001), but also with sialic acid (r = 0.40, P = 0.01) and IL-6 (r = 0.38, P = 0.04). CONCLUSIONS: Serum leptin concentrations in type 2 diabetes are partly related to an acute-phase or stress response, independent of BMI and sex. The association of hyperleptinaemia with elevated serum cortisol provides a mechanism for leptin resistance in type 2 diabetes (glucocorticoids inhibit the central action of leptin). This study provides further support for the theory that type 2 diabetes is asociated with chronic innate immune activation.

Ethnic differences in correlates of microalbuminuria in NIDDM. The role of the acute-phase response.

OBJECTIVE: To investigate whether microalbuminuria is associated with markers of the acute-phase response in NIDDM and whether there are ethnic differences in this association among the three main racial groups in Malaysia. RESEARCH DESIGN AND METHODS: NIDDM patients of Chinese, Indian, and Malay origin attending a diabetic clinic in Kuala Lumpur, Malaysia, were matched for age, sex, diabetes duration, and glycemic control (n = 34 in each group). Urinary albumin-to-creatinine ratio was measured in an early morning urine sample. Biochemical measurements included markers of the acute-phase response: serum sialic acid, triglyceride, and (lowered) HDL cholesterol. RESULTS: The frequency of microalbuminuria did not differ among the Chinese, Indian, and Malay patients (44, 41, and 47%, respectively). In Chinese patients, those with microalbuminuria had evidence of an augmented acute-phase response, with higher serum sialic acid and triglyceride and lower HDL cholesterol levels; and urinary albumin-to-creatinine ratio was correlated with serum sialic acid and triglyceride. The acute-phase response markers were not different in Indians, with microalbuminuria being high in even the normoalbuminuric Indians; only the mean arterial blood pressure was correlated with urinary albumin-to-creatinine ratio in the Indians. Malay NIDDM subjects had an association of microalbuminuria with acute-phase markers, but this was weaker than in the Chinese subjects. CONCLUSIONS: Microalbuminuria is associated with an acute-phase response in Chinese NIDDM patients in Malaysia, as previously found in Caucasian NIDDM subjects. Elevated urinary albumin excretion has different correlates in other racial groups, such as those originating from the Indian subcontinent. The acute-phase response may have an etiological role in microalbuminuria.

NIDDM as a disease of the innate immune system: association of acute-phase reactants and interleukin-6 with metabolic syndrome X.

Non-insulin-dependent diabetes mellitus (NIDDM) is commonly associated with hypertriglyceridaemia, low serum HDL-cholesterol concentrations, hypertension, obesity and accelerated atherosclerosis (metabolic syndrome X). Since a similar dyslipidaemia occurs with the acute-phase response, we investigated whether elevated acute-phase/stress reactants (the innate immune system's response to environmental stress) and their major cytokine mediator (interleukin-6, IL-6) are associated with NIDDM and syndrome X, and may thus provide a unifying pathophysiological mechanism for these conditions. Two groups of Caucasian subjects with NIDDM were studied. Those with any 4 or 5 features of syndrome X (n = 19) were compared with a group with 0 or 1 feature of syndrome X (n = 25) but similar age, sex distribution, diabetes duration, glycaemic control and diabetes treatment. Healthy non-diabetic subjects of comparable age and sex acted as controls. Overnight urinary albumin excretion rate, a risk factor for cardiovascular disease, was also assayed in subjects to assess its relationship to the acute-phase response. Serum sialic acid was confirmed as a marker of the acute-phase response since serum concentrations were significantly related to established acute-phase proteins such as alpha-1 acid glycoprotein (r = 0.82, p < 0.0001). There was a significant graded increase of serum sialic acid, alpha-1 acid glycoprotein, IL-6 and urinary albumin excretion rate amongst the three groups, with the lowest levels in non-diabetic subjects, intermediate levels in NIDDM patients without syndrome X and highest levels in NIDDM patients with syndrome X. C-reactive protein and cortisol levels were also higher in syndrome X-positive compared to X-negative patients and serum amyloid A was higher in both diabetic groups than in the control group. We conclude that NIDDM is associated with an elevated acute-phase response, particularly in those with features of syndrome X. Abnormalities of the innate immune system may be a contributor to the hypertriglyceridaemia, low HDL cholesterol, hypertension, glucose intolerance, insulin resistance and accelerated atherosclerosis of NIDDM. Microalbuminuria may be a component of the acute-phase response.

Serum sialic acid concentration and coronary heart disease in NIDDM.

OBJECTIVE--To examine the association between serum sialic acid concentrations and coronary heart disease (CHD) in a cross-sectional study of non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS--NIDDM patients (n = 145) attending a diabetic clinic were studied. CHD status was assessed by questionnaire and electrocardiogram coding, and potential risk factor assessment included measurement of fasting serum lipid and lipoprotein concentrations, blood pressure, and urinary albumin excretion rate (AER). RESULTS--Male NIDDM patients with CHD had a higher serum sialic acid level than those without CHD: 2.56 (2.24, 2.72) mmol/l vs. 2.24 (2.18, 2.30) mmol/l, P = 0.01, mean (95% confidence interval). They were also older, had a longer duration of diabetes, had a higher AER, had higher total triglyceride, very-low-density lipoprotein triglyceride and cholesterol, and lipoprotein(a) concentrations, and had a lower apolipoprotein A1 concentration. In an age adjusted multiple lipoprotein(a), hypercholesterolemia, and hypertension were associated with CHD. In women, only hypertension treatment was associated with CHD. CONCLUSIONS--There is a strong univariate association between elevated serum sialic acid and CHD in men (but not women) with NIDDM.

Comparison of micro-enzymatic and high-performance liquid chromatographic methods for the assay of serum 1,5-anhydroglucitol.

Serum 1,5-anhydro-D-glucitol (AG) has been proposed as a marker of glycaemic control in diabetic patients. Two methods have been developed which could be applied to routine clinical monitoring of serum AG. We have compared the assay characteristics of an adapted enzyme assay, based on the enzyme pyranose oxidase, with a high-performance anion-exchange chromatographic method using pulsed amperometric detection (HPAEC-PAD). Linearity and minimum detectable concentrations were practically identical (to at least 400 mumol/l and 4 mumol/l AG, respectively), though intra- and inter-assay precision was better with the HPAEC PAD system at a clinically relevant concentration of 40 mumol/l AG (9.8% vs. 11.8% and 9.2% vs. 13.0%, respectively). The recovery of added AG to serum samples was lower with the micro-enzyme assay than the HPAEC-PAD assay (74 +/- 15% vs. 102 +/- 8%). Despite good agreement by linear regression (r = 0.974, P < 0.005), the assay methods did not demonstrate satisfactory agreement when using a difference plot (limits of agreement -16.1 to 18.7 mumol/l). We conclude that although both assay methods are applicable to routine analyses, the HPAEC-PAD is more precise and is more specific for AG than the enzyme based assay.

Plasma sialic acid in animal models of diabetes mellitus: evidence for modulation of sialic acid concentrations by insulin deficiency.

An elevated circulating sialic acid concentration is a risk factor for cardiovascular disease. Serum sialic acid levels are elevated in NIDDM but not in uncomplicated IDDM. To study why sialic acid is increased in some types of diabetes, we assayed plasma sialic acid in various animal models of diabetes: obese (ob/ob) mice, before and after streptozotocin treatment, neonatal streptozotocin-treated (nSTZ) rats, and diabetic BB rats during and after insulin treatment. In obese mice, which exhibit moderate hyperglycemia and marked hyperinsulinemia, plasma sialic acid was decreased by 45% (fed) and 42% (fasted), compared to lean controls. Fasting reduced plasma glucose and insulin but increased sialic acid in the obese and lean mice. There was a negative correlation (r = -0.84, P < 0.001) between log plasma insulin and sialic acid in the lean and obese mice. The plasma sialic acid:globulin ratio was reduced by 35% in obese mice vs. lean controls, indicating that there may be altered sialylation of glycoproteins in obese mice. Streptozotocin treatment of obese and lean mice reduced plasma insulin but increased sialic acid. In nSTZ rats, hyperglycemia was associated with mild hypoinsulinemia, but not significantly different from control animals, and sialic acid was not altered. In diabetic BB rats, plasma glucose rose from a mean of 4.9 to 23.5 mM 48 hr after insulin withdrawal but sialic acid did not change. We conclude that an elevated plasma sialic acid level is associated with marked insulin deficiency, rather than hyperglycemia per se. The magnitude and speed of this change in sialic acid varies between species.

Serum 1,5-anhydro-D-glucitol assay by high performance anion exchange chromatography.

Serum 1,5-anhydroglucitol (AG) concentrations may be an indicator of glycaemic control in diabetic patients. We have therefore developed an HPLC method for assay of serum AG. Serum samples were de-proteinised with trichloroacetic acid and neutralised with the addition of an anion exchange resin. Erythritol was added to serum as an internal standard. For HPLC, CarboPac MA1 ion exchange columns were used, eluting with 80 mmol/l sodium hydroxide solution. Peak detection was by pulsed amperometry using a gold electrode. The serum AG concentration in 37 non-insulin-dependent diabetic patients was significantly lower than in 28 normal subjects: mean and range 19(4-163) vs. 134(92-203) mumol/l, P < 0.001.

Serum sialic acid enzymatic assay based on microtitre plates: application for measuring capillary serum sialic acid concentrations.

We have adapted a commercially-available kit for the enzymatic determination of sialic acid so that the reaction can be carried out in microtitre wells and the coloured end product quantitated on an ELISA plate reader. The small volume of serum used allows sialic acid to be measured in capillary blood samples. The assay was based on release of sialic acid from glycoconjugates by neuraminidase, cleavage of sialic acid by N-acetyl neuraminic acid aldolase to pyruvate, and then oxidation of pyruvate to hydrogen peroxide by pyruvate oxidase. Hydrogen peroxide was determined by the red product formed in the presence of peroxidase, 4-aminoantipyrine and N-ethyl-N-2-hydroxyethyl-3-toluidine. The assay was linear to at least 10 mmol/l and unaffected by haemolysis, and by the addition of glucose, 3-hydroxybutyrate, bilirubin and pyruvate. Capillary serum sialic acid concentrations were not significantly different from simultaneously measured venous serum sialic acid levels. Self-collected capillary blood samples were obtained from healthy subjects over 6 h during the day. No variations in serum sialic acid concentrations were found in response to a meal. We conclude that this micro-adaptation of a specific sialic acid assay will be suitable for epidemiological surveys of serum sialic acid collected by patients and normal subjects.