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Aims: This study aims to evaluate the stability of C-peptide over time and to compare fasting C-peptide and C-peptide response after mixed-meal tolerance test (MMTT) at T90 or T120 with C-peptide area under the curve (AUC) in long-standing type 1 diabetes. Methods: We included 607 type 1 diabetes individuals with diabetes duration >5 years. C-peptide concentrations (ultrasensitive assay) were collected in the fasting state, and in a subpopulation after MMTT (T0, just prior to, T30-T60-T90-T120, 30–120 min after ingestion of mixed-meal) (n = 168). Fasting C-peptide concentrations (in n = 535) at Year 0 and Year 1 were compared. The clinical determinants associated with residual C-peptide secretion and the correspondence of C-peptide at MMTT T90 / T120 and total AUC were assessed. Results: A total of 153 participants (25%) had detectable fasting serum C-peptide (i.e ≥ 3.8 pmol/L). Fasting C-peptide was significantly lower at Year 1 (p < 0.001, effect size = −0.16). Participants with higher fasting C-peptide had a higher age at diagnosis and shorter disease duration and were less frequently insulin pump users. Overall, 109 of 168 (65%) participants had both non-detectable fasting and post-meal serum C-peptide concentrations. The T90 and T120 C-peptide values at MMTT were concordant with total AUC. In 17 (10%) individuals, C-peptide was only detectable at MMTT and not in the fasting state. Conclusions: Stimulated C-peptide was detectable in an additional 10% of individuals compared with fasting in individuals with >5 years of diabetes duration. T90 and T120 MMTT measurements showed good concordance with the MMTT total AUC. Overall, there was a decrease of C-peptide at 1-year follow-up.

Background Not much is known about the effects of glycemic variability (GV) during the pre- and periconception period on pregnancy/perinatal complications. GV could potentially contribute to identification of high-risk pregnancies in women with type 1 diabetes. Methods An explorative retrospective cohort study was conducted between January 2014 and May 2019. Glucose data were retrieved from electronic patient charts. Pre-/periconceptional GV and GV during all three trimesters was expressed as mean glucose, standard deviation (SD), Coefficient of Variation (CV), High Blood Glucose Index (HBGI), Low Blood Glucose Index (LBGI) and Average Daily Risk Range (ADRR). Maternal and neonatal complications were summarized using a composite total complication score. Binary logistic regression analyses were conducted to assess associations between the GV measures and a total complication score>3, a maternal complication score>1 and a neonatal complication score>1. Results Of 63 eligible women, 29 women (38 pregnancies) were included. Women in the group with a total complication score>3 had a significantly higher ADRR at conception (OR 1.1, CI 1.0–1.2, p=0.048). No statistically significant correlations between complication score and any other GV metric besides the ADRR were found. Although not significant, in the group with a complication score>3, odds ratios>1 were found for SD in trimester 1 (OR 1.6, CI 0.6–4.5, p=0.357) and trimester 2 (OR 1.8, CI 0.5–6.2, p=0.376). Conclusions Presence of a positive association between GV and pregnancy and perinatal complications depends on which pregnancy period is assessed and the GV metrics that are used.

Many C-peptide assays are commercially available for research and routine use. However, not all assays yield consistent results, especially in the low concentration ranges. We searched the literature describing C-peptide measurements to assess which assays are mainly used in the diabetes research field and if they are specified. Percentages of publications on C-peptide measurements in type 1 diabetes (T1D), type 2 diabetes (T2D) and other forms of diabetes were 32%, 54% and 14%, respectively. In only 54% of the publications the used assay was specified. Information on detection limit, measurement range and variation was provided in 12%, 2% and 11% of publications, respectively. In 22% of all publications no C-peptides concentrations were mentioned. This may be a problem especially for T1D research, where measuring very low levels of C-peptide is becoming increasingly important and concordance between assays is low.

INTRODUCTION: C-peptide is an important marker to assess residual insulin production in individuals with type 1 diabetes (T1D). The accuracy and detection limits of C-peptide assays are important to detect C-peptide microsecretion and to reliably observe changes over time in these people. We compared and verified two commercially available assays able to measure C-peptide in the picomolar range.METHODS: The ultrasensitive Mercodia enzyme-linked immunosorbent C-peptide assay (ELISA) was compared with the Beckman immunoradiometric assay (IRMA) for C-peptide, assessing reproducibility (coefficient of variation [CV]), limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ).RESULTS: For both assays within-run and between-run variation were high at the low (around the detection limit) C-peptide concentration range, with CVs of around 40%. LoB values for the ultrasensitive ELISA and the IRMA were 1.3 and 0.16 pmol/L respectively. LoD values were 2.4 and 0.54 pmol/L respectively. LoQ values were 9.7 and 3.8 pmol/L respectively. Only the IRMA met the specifications claimed by the manufacturer.CONCLUSIONS: The IRMA provided the lowest threshold for quantification of serum C-peptide. LoQ of commercially available assays should be established in-house before applying them in research studies and clinical trials in which low C-peptide levels have clinical or scientific relevance.

Aim To assess 1) the prevalence of children and adolescents with type 1 diabetes (T1D) changing from low-risk into borderline-high-risk lipid levels or from borderline-high-risk into high-risk lipid levels (‘lose track of lipids’) and 2) the power of a risk score including the determinants HbA1c, body mass index (BMI), gender, age, diabetes duration and ethnicity in predicting which patients lose track of lipids. Methods 651 children and adolescents with T1D were included in this longitudinal retrospective cohort study. Lipid dynamics and the impact of the risk score on losing track of lipids were evaluated. Kaplan-Meier analysis was used to estimate screening intervals. Results 31–43% percent of the patients had lost track of one or more lipids at the next lipid measurement. This happened more frequently in patients with a low-risk lipid level at start. Depending on the lipid parameter, 5% of patients with low-risk lipid levels lost track of lipids after 13–23 months. The risk score based on concomitant information on the determinants was moderately able to predict which patients would lose track of lipids on the short term. Conclusions A considerable number of children and adolescents with T1D loses track of lipids and does so within a 2-year screening interval. The predictive power of a risk score including age, BMI, gender, HbA1c, diabetes duration and ethnicity is only moderate. Future research should focus on another approach to the determinants used in this study or other determinants predictive of losing track of lipids on the short term.

IntroductionThis study aimed to assess the association between fasting serum C-peptide levels and the presence of impaired awareness of hypoglycemia (IAH) in people with type 1 diabetes.Research design and methodsWe performed a cross-sectional study among 509 individuals with type 1 diabetes (diabetes duration 5–65 years). Extensive clinical data and fasting serum C-peptide concentrations were collected and related to the presence or absence of IAH, which was evaluated using the validated Dutch version of the Clarke questionnaire. A multivariable logistic regression model was constructed to investigate the association of C-peptide and other clinical variables with IAH.ResultsIn 129 (25%) individuals, residual C-peptide secretion was detected, while 75 (15%) individuals reported IAH. The median (IQR) C-peptide concentration among all participants was 0.0 (0.0–3.9) pmol/L. The prevalence of severe hypoglycemia was lower in people with demonstrable C-peptide versus those with absent C-peptide (30% vs 41%, p=0.025). Individuals with IAH were older, had longer diabetes duration, more frequently had macrovascular and microvascular complications, and more often used antihypertensive drugs, antiplatelet agents and cholesterol-lowering medication. There was a strong association between IAH and having a severe hypoglycemia in the preceding year. In multivariable regression analysis, residual C-peptide, either continuously or dichotomous, was associated with lower prevalence of IAH (p=0.040–0.042), while age at diabetes onset (p=0.001), presence of microvascular complications (p=0.003) and body mass index (BMI) (p=0.003) were also independently associated with the presence of IAH.ConclusionsHigher BMI, the presence of microvascular complications and higher age at diabetes onset were independent risk factors for IAH in people with type 1 diabetes, while residual C-peptide secretion was associated with lower risk of this complication.

Respiratory chain function becomes less efficient with age resulting in increased levels of damaging reactive oxygen species. We compared rotenone-exposed fibroblast strains from young and old subjects and from offspring of nonagenarian siblings and the partners of the offspring. Rotenone increased reactive oxygen species levels, inhibited growth rate, and increased telomere shortening (all p

Cellular senescence, an important factor in ageing phenotypes, can be induced by replicative exhaustion or by stress. We investigated the relation between maximum replicative capacity, telomere length, stress-induced cellular senescence, and apoptosis/cell death in human primary fibroblast strains obtained from nonagenarians of the Leiden 85-plus Study. Fibroblast strains were cultured until replicative senescence and stressed with rotenone at low passage. Telomere length, senescence-associated-β-galactosidase activity, sub-G1 content, and Annexin-V/PI positivity were measured in nonstressed and stressed conditions. Fibroblast strains with a higher replicative capacity had longer telomeres (p =.054). In nonstressed conditions, replicative capacity was not associated with β-gal activity (p =.07) and negatively with sub-G1 (p =.008). In rotenone-stressed conditions, replicative capacity was negatively associated with β-gal activity (p =.034) and positively with sub-G1 (p =.07). Summarizing, fibroblast strains with a higher maximum replicative capacity have longer telomeres, are less prone to go into stress-induced cellular senescence, and more prone to die after stress.

Unlike various model organisms, cellular responses to stress have not been related to human longevity. We investigated cellular responses to stress in skin fibroblasts that were isolated from young and very old subjects, and from offspring of nonagenarian siblings and their partners, representatives of the general population. Fibroblasts were exposed to rotenone and hyperglycemia and assessed for senescence-associated beta-galactosidase (SA-beta-gal) activity by flow cytometry. Apoptosis/cell death was measured with the Annexin-V/PI assay and cell-cycle analysis (Sub-G1 content) and growth potential was determined by the colony formation assay. Compared with fibroblasts from young subjects, baseline SA-beta-gal activity was higher in fibroblasts from old subjects (P = 0.004) as were stress-induced increases (rotenone: P < 0.001, hyperglycemia: P = 0.027). For measures of apoptosis/cell death, fibroblasts from old subjects showed higher baseline levels (Annexin V+/PI+ cells: P = 0.040, Sub-G1: P = 0.014) and lower stress-induced increases (Sub-G1: P = 0.018) than fibroblasts from young subjects. Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from young subjects (P = 0.017 and 0.006, respectively). Baseline levels of SA-beta-gal activity and apoptosis/cell death were not different between fibroblasts from offspring and partner. Stress-induced increases were lower for SA-beta-gal activity (rotenone: P = 0.064, hyperglycemia: P < 0.001) and higher for apoptosis/cell death (Annexin V+/PI- cells: P = 0.041, Annexin V+/PI+ cells: P = 0.008). Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from offspring (P = 0.001 and 0.024, respectively) whereas rotenone-induced decreases were lower (P = 0.008 and 0.004, respectively). These data provide strong support for the hypothesis that in vitro cellular responses to stress reflect the propensity for human longevity.