Case Report: Off-Label Liraglutide Use in Children With Wolfram Syndrome Type 1: Extensive Characterization of Four Patients

Aims: Wolfram syndrome type 1 is a rare recessive monogenic form of insulin-dependent diabetes mellitus with progressive neurodegeneration, poor prognosis, and no cure. Based on preclinical evidence we hypothesized that liraglutide, a glucagon-like peptide-1 receptor agonist, may be repurposed for the off-label treatment of Wolfram Syndrome type 1. We initiated an off-label treatment to investigate the safety, tolerability, and efficacy of liraglutide in pediatric patients with Wolfram Syndrome type 1.Methods: Pediatric patients with genetically confirmed Wolfram Syndrome type 1 were offered off-label treatment approved by The Regional Network Coordination Center for Rare Diseases, Pharmacological Research IRCCS Mario Negri, and the internal ethics committee. Four patients were enrolled; none refused nor were excluded or lost during follow-up. Liraglutide was administered as a daily subcutaneous injection. Starting dose was 0.3 mg/day. The dose was progressively increased as tolerated, up to the maximum dose of 1.8 mg/day. The primary outcome was evaluating the safety, tolerability, and efficacy of liraglutide in Wolfram Syndrome type 1 patients. Secondary endpoints were stabilization or improvement of C-peptide secretion as assessed by the mixed meal tolerance test. Exploratory endpoints were stabilization of neurological and neuro-ophthalmological degeneration, assessed by optical coherence tomography, electroretinogram, visual evoked potentials, and magnetic resonance imaging.Results: Four patients aged between 10 and 14 years at baseline were treated with liraglutide for 8–27 months. Liraglutide was well-tolerated: all patients reached and maintained the maximum dose, and none withdrew from the study. Only minor transient gastrointestinal symptoms were reported. No alterations in pancreatic enzymes, calcitonin, or thyroid hormones were observed. At the latest follow-up, the C-peptide area under the curve ranged from 81 to 171% of baseline. Time in range improved in two patients. Neuro-ophthalmological and neurophysiological disease parameters remained stable at the latest follow-up.Conclusions: We report preliminary data on the safety, tolerability, and efficacy of liraglutide in four pediatric patients with Wolfram Syndrome type 1. The apparent benefits both in terms of residual C-peptide secretion and neuro-ophthalmological disease progression warrant further studies on the repurposing of glucagon-like peptide-1 receptor agonists as disease-modifying agents for Wolfram Syndrome type 1.

Long-term β-cells autoimmune destruction markers persistence and residual C-peptide secretion in type 1 diabetes mellitus

Backgraund: It believed that autoimmune process maintained only during the first 5 years of diabetes mellitus type 1 (T1D). Recently scientists discovered the high levels of islet autoantibodies (Ab) in long-standing T1D and some of these patients had residual insulin secretion, determined by the level of C-peptide. According to various sources, the prevalence of such observations ranges from 12 to 48%.Aims: The aim of our study was to assess the duration of autoimmune β-cells destruction markers persistence and residual fasting C-peptide secretion in the long-standing T1D, as well as to determine the possible causes and patterns of these processes.Materials and methods: In the study included 237 patients (91 men, 146 women) with T1D. Patients divided in 4 groups, according to disease duration: а — up to 1 year, n=69 (29%); b — 1–5 years, 52 (22%); c — 5–10 years, 57 (24%); d — more than 10 years, 59 (25%). Ab to glutamic acid decarboxylase (GADA), tyrosine phosphatase-like IA-2 (IA2) and zinc T8 (ZnT8A) were detected by Enzyme Immunoassay. Also detected C-peptide levels and retrospectively HbA1с.Results: Antibodies to antigens of β-cell components were detected in 26 (37%) patients in group A, in 17 patients (33%) in group B, in 15 (29%) in group C and in 14 (23%) — G.In the control group (n = 19), an increased level of antibodies was not revealed. Fasting C-peptide levels were as follows: in group «A» — 0.86 ng / ml [0.53; 1.4], «B» — 0.65 ng / ml [0.27; 0.98], « B «- 0.19 ng / ml [0.17; 0.33],» D «- 0.01 ng / ml [0.01; 0.01]. However, in 13 (22%) patients in group D, fasting C-peptide levels were more than 0.09 ng / ml.Conclusion: The data obtained indicate a long-term persistence of markers of the autoimmune process in patients with T1DM. In groups with a long (more than 5 years) course of T1DM, levels of fasting C-peptide more than 30 pmol/L (0.09 ng / ml or 0.03 nmol / L) were noted in 39 (33.6%) cases.

Circulating Hematopoietic (HSC) and Very-Small Embryonic like (VSEL) Stem Cells in Newly Diagnosed Childhood Diabetes type 1 – Novel Parameters of Beta Cell Destruction/Regeneration Balance and Possible Prognostic Factors of Future Disease Course

Aims/Hypothesis We aimed to evaluate hematopoietic stem cells (HSC) and very small embryonic-like stem cells (VSEL) mobilization to establish their role in residual beta cell function maintenance and partial remission occurrence in children newly diagnosed with type 1 diabetes. Methods We recruited 59 type 1 diabetic patients (aged 6–18 years) monitored for 2 years, and 31 healthy children as a control group. HSC and VSEL levels were assessed at disease onset in PBMC isolated from whole peripheral blood with the use of flow cytometry. An assessment of beta cell function was based on C-peptide secretion. Studied groups were stratified on the basis of VSEL, HSC and/or C-peptide median levels in regard to beta cell function and partial remission. Results Patients with higher stimulated C-peptide secretion at disease onset demonstrated lower levels of HSC (p < 0.05), while for VSEL and VSEL/HSC ratio higher values were observed (p < 0.05). Accordingly, after 2 years follow-up, patients with higher C-peptide secretion presented lower initial levels of HSC and higher VSEL/HSC ratio (p < 0.05). Patients with lower values of HSC levels demonstrated a tendency for better partial remission prevalence in the first 3 to 6 months after diagnosis. Conclusions These clinical observations indicate a possible significant role of HSC and VSEL in maintaining residual beta cell function in type 1 diabetic patients. Graphical Abstract

Colonic lactulose fermentation has no impact on glucagon-like peptide-1 and peptide-YY secretion in healthy young men

Context The colon houses most of our gut microbiota, which ferments indigestible carbohydrates. The products of fermentation have been proposed to influence the secretion of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) from the many endocrine cells in the colonic epithelium. However, little is known about the colonic contribution to fasting or postprandial plasma levels of L-cell products. Objective To determine the impact of colonic lactulose fermentation on gut peptide secretion and to evaluate whether colonic endocrine secretion contributes to gut hormone concentrations measurable in the fasting state. Research Design and Methods Ten healthy young men were studied on three occasions after an overnight fast. On two study days, lactulose (20 g) was given orally, and compared to water intake on a third study day. For one of the lactulose visits participants underwent a full colonic evacuation. Over a six-hour study protocol, lactulose fermentation was assessed by measuring exhaled hydrogen (H2), while gut peptide secretion, paracetamol and short chain fatty acid levels were measured in plasma. Results Colonic evacuation markedly reduced hydrogen exhalation after lactulose intake (p=0.013). Our analysis suggests that the colon does not account for the measurable amounts of GLP-1 and PYY present in the circulation during fasting, and that fermentation and peptide secretion are not acutely related. Conclusion Whether colonic luminal contents affect colonic L-cell secretion sufficiently to influence circulating concentrations requires further investigation. Colonic evacuation markedly reduced lactulose fermentation, but hormone releases were unchanged in the present study.

Residual C-peptide secretion and hypoglycemia awareness in people with type 1 diabetes

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.

The sugar-responsive enteroendocrine neuropeptide F regulates lipid metabolism through glucagon-like and insulin-like hormones in Drosophila melanogaster

The enteroendocrine cell (EEC)-derived incretins play a pivotal role in regulating the secretion of glucagon and insulins in mammals. Although glucagon-like and insulin-like hormones have been found across animal phyla, incretin-like EEC-derived hormones have not yet been characterised in invertebrates. Here, we show that the midgut-derived hormone, neuropeptide F (NPF), acts as the sugar-responsive, incretin-like hormone in the fruit fly, Drosophila melanogaster. Secreted NPF is received by NPF receptor in the corpora cardiaca and in insulin-producing cells. NPF-NPFR signalling resulted in the suppression of the glucagon-like hormone production and the enhancement of the insulin-like peptide secretion, eventually promoting lipid anabolism. Similar to the loss of incretin function in mammals, loss of midgut NPF led to significant metabolic dysfunction, accompanied by lipodystrophy, hyperphagia, and hypoglycaemia. These results suggest that enteroendocrine hormones regulate sugar-dependent metabolism through glucagon-like and insulin-like hormones not only in mammals but also in insects.

Peptidylglycine α-amidating monooxygenase as a therapeutic target or biomarker

Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalyzing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumors. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatric abnormalities. To date, inhibitor design has not fully exploited the structures and mechanistic details of PAM.

Tailored generation of insulin producing cells from canine mesenchymal stem cells derived from bone marrow and adipose tissue

The trend of regenerative therapy for diabetes in human and veterinary practices has conceptually been proven according to the Edmonton protocol and animal models. Establishing an alternative insulin-producing cell (IPC) resource for further clinical application is a challenging task. This study investigated IPC generation from two practical canine mesenchymal stem cells (cMSCs), canine bone marrow-derived MSCs (cBM-MSCs) and canine adipose-derived MSCs (cAD-MSCs). The results illustrated that cBM-MSCs and cAD-MSCs contain distinct pancreatic differentiation potential and require the tailor-made induction protocols. The effective generation of cBM-MSC-derived IPCs needs the integration of genetic and microenvironment manipulation using a hanging-drop culture of PDX1-transfected cBM-MSCs under a three-step pancreatic induction protocol. However, this protocol is resource- and time-consuming. Another study on cAD-MSC-derived IPC generation found that IPC colonies could be obtained by a low attachment culture under the three-step induction protocol. Further, Notch signaling inhibition during pancreatic endoderm/progenitor induction yielded IPC colonies through the trend of glucose-responsive C-peptide secretion. Thus, this study showed that IPCs could be obtained from cBM-MSCs and cAD-MSCs through different induction techniques. Also, further signaling manipulation studies should be conducted to maximize the protocol’s efficiency.

Accelerating cryoprotectant diffusion kinetics improves cryopreservation of pancreatic islets

Cryopreservation offers the potential to increase the availability of pancreatic islets for treatment of diabetic patients. However, current protocols, which use dimethyl sulfoxide (DMSO), lead to poor cryosurvival of islets. We demonstrate that equilibration of mouse islets with small molecules in aqueous solutions can be accelerated from > 24 to 6 h by increasing incubation temperature to 37 °C. We utilize this finding to demonstrate that current viability staining protocols are inaccurate and to develop a novel cryopreservation method combining DMSO with trehalose pre-incubation to achieve improved cryosurvival. This protocol resulted in improved ATP/ADP ratios and peptide secretion from β-cells, preserved cAMP response, and a gene expression profile consistent with improved cryoprotection. Our findings have potential to increase the availability of islets for transplantation and to inform the design of cryopreservation protocols for other multicellular aggregates, including organoids and bioengineered tissues.

The sugar-responsive enteroendocrine neuropeptide F regulates lipid metabolism through glucagon-like and insulin-like hormones in Drosophila melanogaster

The enteroendocrine cell (EEC)-derived incretins play a pivotal role in regulating the secretion of glucagon and insulins in mammals. Although glucagon-like and insulin-like hormones have been found across animal phyla, incretin-like EEC-derived hormones have not yet been characterised in invertebrates. Here, we show that the midgut-derived hormone, Neuropeptide F (NPF), acts as the sugar-responsive, an incretin-like hormone in the fruit fly, Drosophila melanogaster. Secreted NPF is received by NPF receptor in the corpora cardiaca and in insulin-producing cells. NPF-NPFR signalling resulted in the suppression of the glucagon-like hormone production and the enhancement of the insulin-like peptide secretion, eventually promoting lipid anabolism. Similar to the loss of incretin function in mammals, loss of midgut NPF led to significant metabolic dysfunction, accompanied by lipodystrophy, hyperphagia, and hypoglycaemia. These results suggest that enteroendocrine hormones regulate sugar-dependent metabolism through glucagon-like and insulin-like hormones not only in mammals but also in insects.