Hydrogen sulfide (H(2)S) is emerging as a physiological neuromodulator as well as a smooth muscle relaxant. We submit the first evidence that blood H(2)S levels are significantly lower in fasting blood obtained from type 2 diabetes patients compared with age-matched healthy subjects, and in streptozotocin-treated diabetic rats compared with control Sprague-Dawley rats. We further observed that supplementation with H(2)S or an endogenous precursor of H(2)S (l-cysteine) in culture medium prevents IL-8 and MCP-1 secretion in high-glucose-treated human U937 monocytes. These first observations led to the hypothesis that lower blood H(2)S levels may contribute to the vascular inflammation seen in diabetes.

Erythrocytes of diabetic patients have abnormal membrane properties. We examined in vivo membrane lipid peroxidation in erythrocytes of diabetic subjects and its possible relationship with hyperglycemia. Lipid peroxidation was assessed in fresh, untreated erythrocytes by quantitating thiobarbituric acid reactivity and an adduct of phospholipids and malonyldialdehyde (MDA), an end product of lipid peroxidation, with thin-layer chromatography of lipid extract of diabetic erythrocytes. There was a significantly increased membrane lipid peroxidation in diabetic erythrocytes compared with nondiabetic erythrocytes. The degree of membrane lipid peroxidative damage in erythrocytes was significantly correlated with the level of glycosylated hemoglobin, an index of mean glucose level for the preceding 3-4 mo. This suggests that peroxidation of membrane lipids and accumulation of MDA occurs in erythrocytes of diabetic patients.

SummaryBackgroundKCNJ11 mutations cause permanent neonatal diabetes through pancreatic ATP-sensitive potassium channel activation. 90% of patients successfully transfer from insulin to oral sulfonylureas with excellent initial glycaemic control; however, whether this control is maintained in the long term is unclear. Sulfonylurea failure is seen in about 44% of people with type 2 diabetes after 5 years of treatment. Therefore, we did a 10-year multicentre follow-up study of a large international cohort of patients with KCNJ11 permanent neonatal diabetes to address the key questions relating to long-term efficacy and safety of sulfonylureas in these patients.MethodsIn this multicentre, international cohort study, all patients diagnosed with KCNJ11 permanent neonatal diabetes at five laboratories in Exeter (UK), Rome (Italy), Bergen (Norway), Paris (France), and Krakow (Poland), who transferred from insulin to oral sulfonylureas before Nov 30, 2006, were eligible for inclusion. Clinicians collected clinical characteristics and annual data relating to glycaemic control, sulfonylurea dose, severe hypoglycaemia, side-effects, diabetes complications, and growth. The main outcomes of interest were sulfonylurea failure, defined as permanent reintroduction of daily insulin, and metabolic control, specifically HbA1c and sulfonylurea dose. Neurological features associated with KCNJ11 permanent neonatal diabetes were also assessed. This study is registered with ClinicalTrials.gov, number NCT02624817.Findings90 patients were identified as being eligible for inclusion and 81 were enrolled in the study and provided long-term (>5·5 years cut-off) outcome data. Median follow-up duration for the whole cohort was 10·2 years (IQR 9·3–10·8). At most recent follow-up (between Dec 1, 2012, and Oct 4, 2016), 75 (93%) of 81 participants remained on sulfonylurea therapy alone. Excellent glycaemic control was maintained for patients for whom we had paired data on HbA1c and sulfonylurea at all time points (ie, pre-transfer [for HbA1c], year 1, and most recent follow-up; n=64)—median HbA1c was 8·1% (IQR 7·2–9·2; 65·0 mmol/mol [55·2–77·1]) before transfer to sulfonylureas, 5·9% (5·4–6·5; 41·0 mmol/mol [35·5–47·5]; p<0·0001 vs pre-transfer) at 1 year, and 6·4% (5·9–7·3; 46·4 mmol/mol [41·0–56·3]; p<0·0001 vs year 1) at most recent follow-up (median 10·3 years [IQR 9·2–10·9]). In the same patients, median sulfonylurea dose at 1 year was 0·30 mg/kg per day (0·14–0·53) and at most recent follow-up visit was 0·23 mg/kg per day (0·12–0·41; p=0·03). No reports of severe hypoglycaemia were recorded in 809 patient-years of follow-up for the whole cohort (n=81). 11 (14%) patients reported mild, transient side-effects, but did not need to stop sulfonylurea therapy. Seven (9%) patients had microvascular complications; these patients had been taking insulin longer than those without complications (median age at transfer to sulfonylureas 20·5 years [IQR 10·5–24·0] vs 4·1 years [1·3–10·2]; p=0·0005). Initial improvement was noted following transfer to sulfo...

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Recent studies have suggested that elevated cellular lipid peroxidation may play a role in the development of cellular dysfunction and other complications of diabetes. People with type 1 diabetes frequently encounter elevated levels of the ketone bodies acetoacetate (AA), beta-hydroxybutyrate (BHB), and acetone (ACE). This study was undertaken to test the hypothesis that ketosis might increase lipid peroxidation and lower glutathione (GSH) levels of red blood cells (RBCs) in diabetic patients. This study demonstrates that incubation of AA with normal RBCs in phosphate-buffered saline (37 degrees C for 24 h) resulted in marked GSH depletion, oxidized glutathione accumulation, hydroxyl radical generation, and increased membrane lipid peroxidation. Increases in oxygen radicals and lipid peroxidation and depletion of GSH in RBCs were not observed with BHB or ACE treatments. Similarly, there was a significant generation of superoxide ion radicals even in a cell-free buffer solution of AA, but not in that of BHB. The presence of BHB together with AA did not influence the capacity of AA to generate oxygen radicals in a cell-free solution or the increase in lipid peroxidation of RBCs incubated with AA. The antioxidants vitamin E and N-acetylcysteine (NAC) blocked increase in lipid peroxidation in AA-treated RBCs. To examine the effects of ketone bodies in vivo, studies were performed that showed a significant decrease in GSH and an increase in lipid peroxidation levels in RBCs of hyperketonemic diabetic patients, but not in normoketonemic type 1 diabetic patients, when compared with age-matched normal subjects. This study demonstrates that elevated levels of the ketone body AA can increase lipid peroxidation and lower GSH levels of RBCs in people with type 1 diabetes.

OBJECTIVE -Diabetic patients have elevated blood levels of interleukin-6 (IL-6), which is known to increase inflammation and the development of vascular disease and atherosclerosis. This study examined the hypothesis that ketosis increases the circulating levels of IL-6 in type 1 diabetic patients as well as the secretion of IL-6 in vitro in a cell culture model using U937 monocytes.RESEARCH DESIGN AND METHODS -Fasting blood was obtained from type 1 diabetic patients and healthy siblings. To examine the effect of ketosis, U937 monocytes were cultured with ketone bodies (acetoacetate [AA], ␤-hydroxybutyrate [BHB]) in the presence or absence of high glucose levels in the medium at 37°C for 24 h. IL-6 was determined by the sandwich enzyme-linked immunosorbent assay method, and intracellular reactive oxygen species (ROS) generation was detected using dihydroethidium dye. RESULTS-The blood level of IL-6 was higher in hyperketonemic (HK) diabetic patients than in normoketonemic (NK) diabetic patients (P Ͻ 0.05) and normal control subjects (P Ͻ 0.05). There was a significant correlation between ketosis and IL-6 levels (r ϭ 0.36, P Ͻ 0.04, n ϭ 34) in the blood of diabetic patients. Cell culture studies found that exogenous addition of the ketone body AA, but not BHB, increases IL-6 secretion and ROS generation in U937 cells. N-acetylcysteine (NAC) prevented the IL-6 secretion in acetoacetate-treated U937 monocytes.CONCLUSIONS -This study demonstrates that hyperketonemia increases IL-6 levels in the blood of type 1 diabetic patients and that NAC can inhibit IL-6 secretion by U937 monocytic cells cultured in a ketotic medium. Diabetes Care 26:2139 -2143, 2003I nterleukin-6 (IL-6), which is secreted by macrophages, lymphocytes, and other cells (1), is an important cytokine that can initiate events leading to atherogenesis by induction of adhesion molecules, monocyte-endothelial interactions, and inflammation injury (1-5). Anti-IL-6 therapy significantly prevents the inflammatory process in mice (6). The role of IL-6 in vascular inflammation has also been shown using IL-6 knockout mice that exhibit resistance to splanchnic artery occlusion shock (6), and in studies (7) that show increased levels of lipid peroxidation and inflammation in mice that overexpress IL-6. This suggests that elevated blood levels of IL-6 are associated with the development of vascular inflammation and atherosclerosis (1,2).IL-6 levels in blood are higher or similar in diabetic patients compared with normal subjects (4,8 -10). Cell culture studies have shown that high glucose concentrations can increase the IL-6 secretion in cultured monocytes (4,11,12). In addition to hyperglycemia, type 1 diabetic patients frequently experience ketosis (hyperketonemia) from excessive fat breakdown because body fuel is derived mainly from fat when the body is in a state of insulin deficiency (13). The blood concentration of ketone bodies (acetoacetate [AA], ␤-hydroxybutyrate [BHB]) may reach 10 mmol/l in patients with severe ketosis, as compared with levels of Ͻ0.5 ...

An elevated blood level of tumor necrosis factor (TNF)-␣ is a validated marker of vascular inflammation, which can result in the development of vascular disease and atherosclerosis. This study examined the hypothesis that ketosis increases the TNF-␣ secretion, both in a cell culture model using U937 monocytes and in type 1 diabetic patients in vivo. U937 cells were cultured with ketone bodies (acetoacetate [AA] and ␤-hydroxybutyrate [BHB]) in the presence or absence of high levels of glucose in medium at 37°C for 24 h. This study demonstrates the following points. First, hyperketonemic diabetic patients have significantly higher levels of TNF-␣ than normoketonemic diabetic patients (P < 0.01) and normal control subjects (P < 0.01). There was a significant correlation (r ‫؍‬ 0.36, P < 0.05; n ‫؍‬ 34) between ketosis and oxidative stress as well as between oxidative stress and TNF-␣ levels (r ‫؍‬ 0.47, P < 0.02; n ‫؍‬ 34) in the blood of diabetic patients. Second,

OBJECTIVE -This study examined the relationship between cellular glutathione and vitamin E concentrations and the effect of vitamin E (␣-tocopherol) supplementation on glutathione and lipid peroxidation product concentrations in the erythrocytes of type 1 diabetic patients.RESEARCH DESIGN AND METHODS -We obtained written informed consent to participate in this study from diabetic patients (n = 29) and their age-matched nondiabetic siblings (n = 21) according to the guidelines of the Institutional Review Board on Human Experimentation. Diabetic patients were supplemented with a DL-␣-tocopherol (vitamin E) capsule (100 IU/ orally) or placebo for 3 months in a double-blind clinical trial. Fasting blood samples were collected from each diabetic patient before the start of and after the 3 months of vitamin E or placebo supplementation. Glutathione, malondialdehyde (which is a product of lipid peroxidation), and ␣-tocopherol were determined using high-performance liquid chromatography. A total of 5 diabetic patients were excluded after randomization from the data analyses. Data were analyzed statistically using a paired Student' s t test to compare 12 diabetic patients taking vitamin E with 12 diabetic patients receiving placebo supplementation and to compare diabetic patients with healthy nondiabetic subjects.RESULTS -Erythrocytes of diabetic patients had 21% higher (P Ͻ 0.001) malondialdehyde and 15% lower (P Ͻ 0.05) glutathione concentrations than healthy subjects. Vitamin E in erythrocytes had a significant correlation with the glutathione concentrations in the erythrocytes (r = 0.46, P Ͻ 0.02). Vitamin E supplementation increased glutathione concentrations by 9% (P Ͻ 0.01) and lowered concentrations of malondialdehyde by 23% (P Ͻ 0.001) and of HbA 1c by 16% (P Ͻ 0.02) in erythrocytes of diabetic patients. No differences were evident in these parameters before versus after placebo supplementation.CONCLUSIONS -Glutathione level is significantly related to vitamin E level, and supplementation with vitamin E (100 IU/day) significantly increases glutathione and lowers lipid peroxidation and HbA 1c concentrations in the erythrocytes of type 1 diabetic patients.