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Aims/hypothesis Diabetic gastroenteropathy frequently causes debilitating gastrointestinal symptoms. Previous uncontrolled studies have shown that transcutaneous vagal nerve stimulation (tVNS) may improve gastrointestinal symptoms. To investigate the effect of cervical tVNS in individuals with diabetes suffering from autonomic neuropathy and gastrointestinal symptoms, we conducted a randomised, sham-controlled, double-blind (participants and investigators were blinded to the allocated treatment) study. Methods This study included adults (aged 20–86) with type 1 or 2 diabetes, gastrointestinal symptoms and autonomic neuropathy recruited from three Steno Diabetes Centres in Denmark. Participants were randomly allocated 1:1 to receive active or sham stimulation. Active cervical tVNS or sham stimulation was self-administered over two successive study periods: 1 week of four daily stimulations and 8 weeks of two daily stimulations. The primary outcome measures were gastrointestinal symptom changes as measured using the gastroparesis cardinal symptom index (GCSI) and the gastrointestinal symptom rating scale (GSRS). Secondary outcomes included gastrointestinal transit times and cardiovascular autonomic function. Results Sixty-eight participants were randomised to the active group, while 77 were randomised to the sham group. Sixty-three in the active and 68 in the sham group remained for analysis in study period 1, while 62 in each group were analysed in study period 2. In study period 1, active and sham tVNS resulted in similar symptom reductions (GCSI: −0.26 ± 0.64 vs −0.17 ± 0.62, p=0.44; GSRS: −0.35 ± 0.62 vs −0.32 ± 0.59, p=0.77; mean ± SD). In study period 2, active stimulation also caused a mean symptom decrease that was comparable to that observed after sham stimulation (GCSI: −0.47 ± 0.78 vs −0.33 ± 0.75, p=0.34; GSRS: −0.46 ± 0.90 vs −0.35 ± 0.79, p=0.50). Gastric emptying time was increased in the active group compared with sham (23, Aims/hypothesis: Diabetic gastroenteropathy frequently causes debilitating gastrointestinal symptoms. Previous uncontrolled studies have shown that transcutaneous vagal nerve stimulation (tVNS) may improve gastrointestinal symptoms. To investigate the effect of cervical tVNS in individuals with diabetes suffering from autonomic neuropathy and gastrointestinal symptoms, we conducted a randomised, sham-controlled, double-blind (participants and investigators were blinded to the allocated treatment) study. Methods: This study included adults (aged 20–86) with type 1 or 2 diabetes, gastrointestinal symptoms and autonomic neuropathy recruited from three Steno Diabetes Centres in Denmark. Participants were randomly allocated 1:1 to receive active or sham stimulation. Active cervical tVNS or sham stimulation was self-administered over two successive study periods: 1 week of four daily stimulations and 8 weeks of two daily stimulations. The primary outcome measures were gastrointestinal symptom changes as measured using the gastroparesis cardinal symptom index (GCSI) and the gastrointestinal symptom rating scale (GSRS). Secondary outcomes included gastrointestinal transit times and cardiovascular autonomic function. Results: Sixty-eight participants were randomised to the active group, while 77 were randomised to the sham group. Sixty-three in the active and 68 in the sham group remained for analysis in study period 1, while 62 in each group were analysed in study period 2. In study period 1, active and sham tVNS resulted in similar symptom reductions (GCSI: −0.26 ± 0.64 vs −0.17 ± 0.62, p=0.44; GSRS: −0.35 ± 0.62 vs −0.32 ± 0.59, p=0.77; mean ± SD). In study period 2, active stimulation also caused a mean symptom decrease that was comparable to that observed after sham stimulation (GCSI: −0.47 ± 0.78 vs −0.33 ± 0.75, p=0.34; GSRS: −0.46 ± 0.90 vs −0.35 ± 0.79, p=0.50). Gastric emptying time was increased in the active group compared with sham (23 min vs −19 min, p=0.04)

Background: Diabetic cardiovascular autonomic neuropathy (CAN) and distal symmetrical polyneuropathy (DSPN) are severe diabetic complications. Collagen type VI (COL6) and III (COL3) have been associated with nerve function. We investigated if markers of COL6 formation (PRO-C6) and COL3 degradation (C3M) were associated with neuropathy in people with type 1 diabetes (T1D). Methods: In a cross-sectional study including 300 people with T1D, serum and urine PRO-C6 and C3M were obtained. CAN was assessed by cardiovascular reflex tests: heart rate response to deep breathing (E/I ratio), to standing (30/15 ratio) and to the Valsalva maneuver (VM). Two or three pathological CARTs constituted CAN. DSPN was assessed by biothesiometry. Symmetrical vibration sensation threshold above 25 V constituted DSPN. Results: Participants were (mean (SD)) 55.7 (9.3) years, 51% were males, diabetes duration was 40.0 (8.9) years, HbA1c was 63 (11 mmol/mol, (median (IQR)) serum PRO-C6 was 7.8 (6.2;11.0) ng/ml and C3M 8.3 (7.1;10.0) ng/ml. CAN and DSPN were diagnosed in 34% and 43% of participants, respectively. In models adjusted for relevant confounders a doubling of serum PRO-C6, was significantly associated with odds ratio > 2 for CAN and > 1 for DSPN, respectively. Significance was retained after additional adjustments for eGFR only for CAN. Higher serum C3M was associated with presence of CAN, but not after adjustment for eGFR. C3M was not associated with DSPN. Urine PRO-C6 analyses indicated similar associations. Conclusions: Results show previously undescribed associations between markers of collagen turnover and risk of CAN and to a lesser degree DSPN in T1D.

This study aims to examine the association between baseline level and change of autonomic nervous function with subsequent development of arterial stiffness. Autonomic nervous function was assessed in 4901 participants of the Whitehall II occupational cohort by heart rate variability (HRV) indices and resting heart rate (rHR) three times between 1997 and 2009, while arterial stiffness was assessed by carotid-femoral pulse wave velocity (PWV) measured twice between 2007 and 2013. First, individual HRV/rHR levels and annual changes were estimated. Then, we modelled the development of PWV by HRV/rHR using linear mixed effect models. First, we adjusted for sex and ethnicity (model 1), and then for socioeconomic and lifestyle factors, various clinical measurements, and medications (model 2). A decrease in HRV and unchanged rHR was associated with subsequent higher levels of PWV, but the effect of a change in HRV was less pronounced at higher ages. A typical individual aged 65 years with a SDNN level of 30 ms and a 2% annual decrease in SDNN had 1.32 (0.95; 1.69) higher PWV compared to one with the same age and SDNN level but with a 1% annual decrease in SDNN. Further adjustment had no major effect on the results. People who experience a steeper decline in autonomic nervous function have higher levels of arterial stiffness. The association was stronger in younger people., This study aims to examine the association between baseline level and change of autonomic nervous function with subsequent development of arterial stiffness. Autonomic nervous function was assessed in 4901 participants of the Whitehall II occupational cohort by heart rate variability (HRV) indices and resting heart rate (rHR) three times between 1997 and 2009, while arterial stiffness was assessed by carotid-femoral pulse wave velocity (PWV) measured twice between 2007 and 2013. First, individual HRV/rHR levels and annual changes were estimated. Then, we modelled the development of PWV by HRV/rHR using linear mixed effect models. First, we adjusted for sex and ethnicity (model 1), and then for socioeconomic and lifestyle factors, various clinical measurements, and medications (model 2). A decrease in HRV and unchanged rHR was associated with subsequent higher levels of PWV, but the effect of a change in HRV was less pronounced at higher ages. A typical individual aged 65 years with a SDNN level of 30 ms and a 2% annual decrease in SDNN had 1.32 (0.95; 1.69) higher PWV compared to one with the same age and SDNN level but with a 1% annual decrease in SDNN. Further adjustment had no major effect on the results. People who experience a steeper decline in autonomic nervous function have higher levels of arterial stiffness. The association was stronger in younger people.

OBJECTIVE: We investigated the association between the cardiovascular autonomic neuropathy (CAN) diagnosis and glucose variability (GV) in type 1 diabetes (T1D), as autonomic dysfunction previously has been associated with increased GV.RESEARCH DESIGN AND METHODS: CAN was assessed by three recommended cardiovascular reflex tests (CARTs). Glucose metrics were obtained from 10-day blinded continuous glucose monitoring (CGM). Between-group differences in GV indices were assessed by regression analyses in 24 participants with T1D with CAN and 24 matched control subjects without CAN.RESULTS: The CAN diagnosis was associated with 4.9% (95% CI 1.0, 8.7) higher coefficient of variation (CV) (P = 0.014), 0.7 mmol/L (0.3, 1.1) higher SD (P = 0.002) of glucose, and 1.4 mmol/mol (0.0, 2.7) higher mean amplitude of glycemic excursions (P = 0.047). Lower measures of CARTs were associated with higher CV, SD, and time above range values.CONCLUSIONS: The CAN diagnosis associates with a significantly higher GV in T1D, despite a high prevalence of routine CGM use.

Introduction: Diabetic cardiovascular autonomic neuropathy (CAN) is associated with increased mortality and morbidity. To explore metabolic mechanisms associated with CAN we investigated associations between serum metabolites and CAN in persons with type 1 diabetes (T1D). Materials and Methods: Cardiovascular reflex tests (CARTs) (heart rate response to: deep breathing; lying-to-standing test; and the Valsalva maneuver) were used to diagnose CAN in 302 persons with T1D. More than one pathological CARTs defined the CAN diagnosis. Serum metabolomics and lipidomic profiles were analyzed with two complementary non-targeted mass-spectrometry methods. Cross-sectional associations between metabolites and CAN were assessed by linear regression models adjusted for relevant confounders. Results: Participants were median (IQR) aged 55(49, 63) years, 48% males with diabetes duration 39(32, 47) years, HbA1c 63(55,69) mmol/mol and 34% had CAN. A total of 75 metabolites and 106 lipids were analyzed. In crude models, the CAN diagnosis was associated with higher levels of hydroxy fatty acids (2,4- and 3,4-dihydroxybutanoic acids, 4−deoxytetronic acid), creatinine, sugar derivates (ribitol, ribonic acid, myo-inositol), citric acid, glycerol, phenols, phosphatidylcholines and lower levels of free fatty acids and the amino acid methionine (p<0.05). Upon adjustment, positive associations with the CAN diagnoses were retained for hydroxy fatty acids, tricarboxylic acid (TCA) cycle-based sugar derivates, citric acid, and phenols (P<0.05). Conclusion: Metabolic pathways, including the TCA cycle, hydroxy fatty acids, phosphatidylcholines and sugar derivatives are associated with the CAN diagnosis in T1D. These pathway may be part of the pathogeneses leading to CAN and may be modifiable risk factors for the complication.

Background: The mechanisms linking cardiovascular autonomic neuropathy, diabetic kidney disease and cardiovascular mortality in type 2 diabetes are widely unknown. We investigated the relationship between baseline cardiovascular autonomic function and changes in kidney and myocardial function over six years in patients with type 2 diabetes and healthy controls.Methods: Post-hoc analysis of a cohort study in 24 patients with type 2 diabetes and 18 healthy controls. Baseline determinants were cardiovascular autonomic reflex tests (heart rate response to: standing (30:15); deep breathing (E:I); and the Valsalva test) and time- and frequency-domain heart rate variability indices. Outcomes were changes in estimated glomerular filtration rate (eGFR), albuminuria, myocardial flow reserve (MFR) measured by cardiac 82Rb Positron emission tomography computed tomography (PET/CT), and coronary artery calcium score (CACS).Results: Mean age at inclusion was 61 ± 10 years and 36% were female. Mean follow up time was 6 ± 0 years. A lower response in heart rate to the Valsalva test (corresponding to weaker autonomic function) was associated with a larger decline in eGFR (p=0.04), but not significantly after adjustment for sex, baseline age, smoking status, systolic blood pressure, heart rate, HbA1c, body mass index and baseline eGFR (p=0.12). A higher baseline response in heart rate to standing (30:15) was associated with a larger decline in myocardial flow reserve in the unadjusted analysis (p=0.02) and after adjustment (p=0.02). A higher response in heart rate to the Valsalva maneuver was associated with a larger increase in CACS (p = 0.02), but the association became insignificant after adjustment (p = 0.16).Conclusion: A lower response in heart rate to the Valsalva test was associated with a larger decline in kidney function, indicating that autonomic dysfunction may predict future loss of kidney function. However, we did not find any association between

Background: Microalbuminuria is an early sign of kidney disease in people with diabetes and indicates increased risk of cardiovascular disease. We tested whether a urinary proteomic risk classifier (CKD273) score was associated with development of microalbuminuria and whether progression to microalbuminuria could be prevented with the mineralocorticoid receptor antagonist spironolactone. Methods: In this multicentre, prospective, observational study with embedded randomised controlled trial (PRIORITY), we recruited people with type 2 diabetes, normal urinary albumin excretion, and preserved renal function from 15 specialist centres in ten European countries. All participants (observational cohort) were tested with the CKD273 classifier and classified as high risk (CKD273 classifier score >0·154) or low risk (≤0·154). Participants who were classified as high risk were entered into a randomised controlled trial and randomly assigned (1:1), by use of an interactive web-response system, to receive spironolactone 25 mg once daily or matched placebo (trial cohort). The primary endpoint was development of confirmed microalbuminuria in all individuals with available data (observational cohort). Secondary endpoints included reduction in incidence of microalbuminuria with spironolactone (trial cohort, intention-to-treat population) and association between CKD273 risk score and measures of impaired renal function based on estimated glomerular filtration rate (eGFR; observational cohort). Adverse events (particularly gynaecomastia and hyperkalaemia) and serious adverse events were recorded for the intention-to-treat population (trial cohort). This study is registered with the EU Clinical Trials Register (EudraCT 20120-004523-4) and ClinicalTrials.gov (NCT02040441) and is completed. Findings: Between March 25, 2014, and Sept 30, 2018, we enrolled and followed-up 1775 participants (observational cohort), 1559 (88%) of 1775 participants had a low-risk urinary proteomic patte

Background-Peripheral artery disease is a major socioeconomic challenge in the diabetes mellitus community and non-surgical treatment options are limited. As remote ischemic conditioning (RIC) improves vascular function and attenuates ischemia-induced tissue damage, we investigated the efficacy of RIC on vascular and neuronal function in type 2 diabetes mellitus patients with peripheral artery disease. Methods and Results-We enrolled 36 type 2 diabetes mellitus patients with moderately reduced toe pressure (40-70 mm Hg) in a randomized sham-controlled double-masked trial. Patients were allocated to 12 weeks once daily upper arm cuff-based treatment of either RIC treatment (4 cycles of 5-minute ischemia followed by 5-minute reperfusion) or similar sham-device treatment. Primary outcome was transcutaneous tissue oxygen tension of the instep of the feet. Secondary outcomes were aortic pulse wave velocity, toe pressure and toe-brachial index. Tertiary outcomes were markers of peripheral and autonomic nerve function. We enrolled 36 patients (83% men). Patients had a mean (SD) age of 70.7 years (6.8), diabetes mellitus duration of 18.4 years (8.3), HbA1c (gycated hemoglobin) of 59.7 mmol/mol (11.2). Eighty percent had peripheral symmetrical neuropathy. The mean difference in change of transcutaneous tissue oxygen tension from baseline between the RIC and sham-treated groups was -0.03 mm Hg ([95% CI -0.1; 0.04], P=0.438). RIC did not elicit any change in additional outcomes. Three patients experienced transient skin petechiae in the treated arm. Conclusions-Long-term repeated remote ischemic conditioning treatment have no effect on tissue oxygenation, vascular or neuronal function in patients with type 2 diabetes mellitus and moderate peripheral artery disease.

The aim of this study was to investigate the association of epicardial (EAT) and pericardial (PAT) adipose tissues with myocardial function in type 2 diabetes (T2D). EAT and PAT were measured by ultrasound in 770 patients with T2D and 234 age- and sex-matched non-diabetic controls. Echocardiography was performed, including tissue Doppler imaging and 2D speckle tracking. Patients with T2D versus controls had increased EAT (4.6 ± 1.8 mm vs. 3.4 ± 1.2 mm, P < 0.0001) and PAT (6.3 ± 2.8 mm vs. 5.3 ± 2.4 mm, P < 0.0001). EAT and PAT were associated with structural cardiac measures both in T2D patients and controls (all P < 0.043), but only in T2D patients with functional measures: PAT was associated with impaired global longitudinal strain [beta coefficient (SE)] [0.11% (0.04), P = 0.002], while EAT was associated with reduced diastolic function by lateral early diastolic myocardial velocity (e’lat) [−0.31 (0.05) cm/s, P = 0.001], mitral inflow velocities: peak early (E)/peak atrial (A) ratio [−0.02 (0.01), P = 0.001] and lateral E/e’lat [0.36 (0.10), P < 0.001]. However, no interaction was found between diabetes status and PAT (P = 0.75) or EAT (P = 0.45). Adipose tissue in intimate relation to the myocardium is higher in patients with T2D versus controls and is associated with functional myocardial measures in T2D.

AIMS: Vitamin B12 deficiency could be associated with cardiovascular autonomic neuropathy (CAN) in diabetes patients. We aim to investigate the association between serum levels of vitamin B12 and CAN in type 2 diabetes patients.METHODS: 469 ambulatory type 2 diabetes patients (mean diabetes duration 10.0years (IQR 5.0;17.0), mean age 59.0years (SD 11.6), 63% men, mean B12 289.0pmol/l (IQR 217;390)) were screened for CAN using three cardiovascular reflex tests, five minute resting heart rate (5min RHR) and heart rate variability indices.RESULTS: Serum levels of vitamin B12 were significantly lower in patients treated with metformin and/or proton pump inhibitors (PPIs) compared with patients not treated (p<0.001). A 25pmol/l higher level of vitamin B12 was associated with an odds ratio of the CAN diagnosis of 0.94 (95% CI 0.88; 1.00, p=0.034), an increase in E/I-ratio of 0.21% (95% CI 0.01; 0.43, p=0.038), and a decrease in 5min RHR of 0.25 beats per minute (95% CI -0.47; -0.03, p=0.025).CONCLUSION: Vitamin B12 may be inversely associated with CAN in patients with type 2 diabetes. Confirmatory studies investigating a causal role of vitamin B12 for the development of diabetic CAN are warranted.

Background We evaluated the association of cardiac adipose tissue including epicardial adipose tissue and pericardial adipose tissue with incident cardiovascular disease and mortality, coronary artery calcium, carotid intima media thickness and inflammatory markers. Design A prospective study of 200 patients with type 2 diabetes and elevated urinary albumin excretion rate (UAER). Methods Cardiac adipose tissue was measured from baseline echocardiography. The composite endpoint comprised incident cardiovascular disease and all-cause mortality. Coronary artery calcium, carotid intima media thickness and inflammatory markers were measured at baseline. Cardiac adipose tissue was investigated as continuous and binary variable. Analyses were performed unadjusted (model 1), and adjusted for age, sex (model 2), body mass index, low-density lipoprotein cholesterol, smoking, glycated haemoglobin, and systolic blood pressure (model 3). Results Patients were followed-up after 6.1 years for non-fatal cardiovascular disease (n = 29) or mortality (n = 23). Cardiac adipose tissue (p = 0.049) and epicardial adipose tissue (p = 0.029) were associated with cardiovascular disease and mortality in model 1. When split by the median, patients with high cardiac adipose tissue had a higher risk of cardiovascular disease and mortality than patients with low cardiac adipose tissue in unadjusted (hazard ratio 1.9, confidence interval: 1.1; 3.4, p = 0.027) and adjusted (hazard ratio 2.0, confidence interval: 1.1; 3.7, p = 0.017) models. Cardiac adipose tissue (p = 0.033) was associated with baseline coronary artery calcium (model 1) and interleukin-8 (models 1-3, all p < 0.039). Conclusions In type 2 diabetes patients without coronary artery disease, high cardiac adipose tissue levels were associated with increased risk of incident cardiovascular disease or all-cause mortality even after accounting for traditional cardiovascular disease risk factors. High cardiac adipose tissue amounts we

Snakebite envenoming is a serious condition requiring medical attention and administration of antivenom. Current antivenoms are antibody preparations obtained from the plasma of animals immunised with whole venom(s) and contain antibodies against snake venom toxins, but also against other antigens. In order to better understand the molecular interactions between antivenom antibodies and epitopes on snake venom toxins, a high-throughput immuno-profiling study on all manually curated toxins from Dendroaspis species and selected African Naja species was performed based on custom-made high-density peptide microarrays displaying linear toxin fragments. By detection of binding for three different antivenoms and performing an alanine scan, linear elements of epitopes and the positions important for binding were identified. A strong tendency of antivenom antibodies recognizing and binding to epitopes at the functional sites of toxins was observed. With these results, high-density peptide microarray technology is for the first time introduced in the field of toxinology and molecular details of the evolution of antibody-toxin interactions based on molecular recognition of distinctive toxic motifs are elucidated., Snakebite envenoming is a serious condition requiring medical attention and administration of antivenom. Current antivenoms are antibody preparations obtained from the plasma of animals immunised with whole venom(s) and contain antibodies against snake venom toxins, but also against other antigens. In order to better understand the molecular interactions between antivenom antibodies and epitopes on snake venom toxins, a high-throughput immuno-profiling study on all manually curated toxins from Dendroaspis species and selected African Naja species was performed based on custom-made high-density peptide microarrays displaying linear toxin fragments. By detection of binding for three different antivenoms and performing an alanine scan, linear elements of epitopes and the positions important for binding were identified. A strong tendency of antivenom antibodies recognizing and binding to epitopes at the functional sites of toxins was observed. With these results, high-density peptide microarray technology is for the first time introduced in the field of toxinology and molecular details of the evolution of antibody-toxin interactions based on molecular recognition of distinctive toxic motifs are elucidated.