Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing.

The loss of first-phase insulin secretion is a characteristic feature of type 2 diabetic patients. The fast-acting insulin analog lispro provides a therapeutic tool for assessing the metabolic outcome of restoration of an early rise in plasma insulin levels after the ingestion of an oral glucose load. We studied eight type 2 diabetic patients on two different occasions when they received an oral glucose load (50 g) preceded by either human regular insulin or insulin analog lispro (both 0.075 U/kg lean body mass). Tritiated glucose was infused throughout the studies, and the oral glucose was labeled with [13C6]glucose for monitoring systemic and oral glucose kinetics, respectively. Basal plasma glucose (8.2 +/- 0.9 vs. 7.5 +/- 0.8 mmol/l), insulin (224 +/- 21 vs. 203 +/- 21 pmol/l), and endogenous glucose production (10.4 +/- 1.0 vs. 11.1 +/- 1.1 micromol x kg(-1) x min(-1)) were similar on both occasions. In spite of comparable incremental areas under the curve, the time course of plasma insulin concentration was much different. After injection of regular insulin, plasma insulin peaked at 120 min (368 +/- 42 pmol/l), while with lispro, the peak occurred at 60 min (481 +/- 42 pmol/l). Plasma insulin concentration during the last 3 h of the study, however, was lower with lispro compared with regular insulin. The incremental area under the curve of plasma C-peptide was lower with lispro (0.05 +/- 0.01 vs. 0.13 +/- 0.04 micromol/300 min; P < 0.01). After the ingestion of the oral glucose load, plasma glucose concentration increased by 78% at 80-100 min with regular insulin and by 62% with lispro (P < 0.05) and remained lower for the ensuing 3 h. The incremental area under the curve was 46% lower with lispro (715 +/- 109 vs. 389 +/- 109 pmol/300 min; P < 0.01). There was no difference in the two studies in the rate of appearance of the ingested glucose and in the overall rate of glucose disposal. During the initial 90 min, however, the rate of endogenous glucose production was suppressed in a prompter and more profound manner when lispro was administered (1.39 +/- 0.10 vs. 5.00 +/- 1.22 micromol x kg(-1) x min(-1); P < 0.05), while there was no difference in the late prandial phase. These results show that an early rise in plasma insulin levels after the ingestion of a glucose load is associated with a significant improvement in glucose tolerance due to a prompter, though short-lived, suppression of endogenous glucose production. This amelioration in plasma glucose profile prevents late hyperglycemia and hyperinsulinemia. Therefore, restoration of a more physiologic profile of prandial plasma insulin profile represents a rational approach for treatment of type 2 diabetic patients.

New effort has been made to develop closed-loop glucose control, using subcutaneous (SC) glucose sensing and continuous subcutaneous insulin infusion (CSII) from a pump, and a control algorithm. An approach based on a model predictive control (MPC) algorithm has been utilized during closed-loop control in type 1 diabetes patients. Here we describe the preliminary clinical experience with this approach. Six type 1 diabetes patients (three in each of two clinical investigation centers in Padova and Montpellier), using CSII, aged 36 +/- 8 and 48 +/- 6 years, duration of diabetes 12 +/- 8 and 29 +/- 4 years, hemoglobin A1c 7.4% +/- 0.1% and 7.3% +/- 0.3%, body mass index 23.2 +/- 0.3 and 28.4 +/- 2.2 kg/m(2), respectively, were studied on two occasions during 22 h overnight hospital admissions 2-4 weeks apart. A Freestyle Navigator(R) continuous glucose monitor and an OmniPod insulin pump were applied in each trial. Admission 1 used open-loop control, while admission 2 employed closed-loop control using our MPC algorithm. In Padova, two out of three subjects showed better performance with the closed-loop system compared to open loop. Altogether, mean overnight plasma glucose (PG) levels were 134 versus 111 mg/dl during open loop versus closed loop, respectively. The percentage of time spent at PG > 140 mg/dl was 45% versus 12%, while postbreakfast mean PG was 165 versus 156 mg/dl during open loop versus closed loop, respectively. Also, in Montpellier, two patients out of three showed a better glucose control during closed-loop trials. Avoidance of nocturnal hypoglycemic excursions was a clear benefit during algorithm-guided insulin delivery in all cases. This preliminary set of studies demonstrates that closed-loop control based entirely on SC glucose sensing and insulin delivery is feasible and can be applied to improve glucose control in patients with type 1 diabetes, although the algorithm needs to be further improved to achieve better glycemic control.

The effects of physiologic hyperinsulinemia and hyperaminoacidemia, alone or in combination, on leucine kinetics in vivo were studied in postabsorptive healthy subjects with primed-constant infusions of L-[4,5-3Hjleucine and I1-14Cqa-ketoisocaproate (KIC) under euglycemic conditions. Hyperinsulinemia (-100 ,U/ml) decreased (P < 0.05 vs. baseline) steady state Leucine + KIC rates of appearance (Ra) from proteolysis, KIC (-leucine-carbon) oxidation, and nonoxidized leucine-carbon flux (leucine protein). Hyperaminoacidemia (plasma leucine, 210 pimol/liter), with either basal hormone replacement or combined to hyperinsulinemia, resulted in comparable increases in leucine + KIC Ra, KIC oxidation, and leucine --protein (P < 0.05 vs. baseline).However, endogenous leucine + KIC Ra was suppressed only with the combined infusion. Therefore, on the basis of leucine kinetic data, hyperinsulinemia and hyperaminoacidemia stimulated net protein anabolism in vivo by different mechanisms. Hyperinsulinemia decreased proteolysis but did not stimulate leucine --protein. Hyperaminoacidemia per se stimulated leucine --protein but did not suppress endogenous proteolysis.When combined, they had a cumulative effect on net leucine deposition into body protein.

BackgroundSodium-glucose co-transporter-2 inhibitors (SGLT2i) reduce glucose levels, body weight, and blood pressure, possibly resulting in cardiovascular protection. In phase III trials, SGLT2i were shown to increase HDL cholesterol. We aimed to evaluate whether the SGLT2i dapagliflozin affects HDL function in a randomized placebo-controlled trial.MethodsThirty-three type 2 diabetic patients were randomized to receive dapagliflozin 10 mg or placebo for 12 weeks on top of their glucose lowering medications. The primary end-point was the change in cholesterol efflux capacity (CEC) from macrophages at study end versus baseline. Secondary endpoints were changes in: distribution of HDL subfractions, lipid profile, activity of enzymes that mediate HDL antioxidant properties (PON1 and ARE) and cholesterol metabolism (CETP), HbA1c, body weight and composition.ResultsThirty-one patients completed the study, n = 16 in the placebo group and n = 15 in the dapagliflozin group. Patients randomized to dapagliflozin were older and had lower adiposity indexes, although these differences disappeared after correction for multiple testing. Therapy with dapagliflozin reduced HbA1c by 0.9% and body weight by 3.1 kg, mainly attributable to reduction of body water and lean mass. As compared to placebo, dapagliflozin reduced CEC (−6.7 ± 2.4 versus 0.3 ± 1.8%; p = 0.043), but this effect was no longer significant after adjusting for age and BMI. No change was detected in HDL cholesterol, HDL subfractions, activity of PON1, ARE, and CETP.ConclusionsDespite improvements in glucose control and reduction in body weight, therapy with dapagliflozin exerted no significant effect on HDL cholesterol levels and HDL functionality. Trial registration EudraCT 2014-004270-42; NCT02327039Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-017-0529-3) contains supplementary material, which is available to authorized users.