Diabetologia -

Aims/hypothesis Type 1 diabetes manifests after irreversible beta cell damage, highlighting the crucial need for markers of the presymptomatic phase to enable early and effective interventions. Current efforts to identify molecular markers of disease-triggering events lack resolution and convenience. Analysing frequently self-collected dried blood spots (DBS) could enable the detection of early disease-predictive markers and facilitate tailored interventions. Here, we present a novel strategy for monitoring transient molecular changes induced by environmental triggers that enable timely disease interception. Methods Whole blood (10 μl) was sampled regularly (every 1–5 days) from adult NOD mice infected with Coxsackievirus B3 (CVB3) or treated with vehicle alone. Blood samples (5 μl) were dried on filter discs. DBS samples were analysed by proximity extension assay. Generalised additive models were used to assess linear and non-linear relationships between protein levels and the number of days post infection (p.i.). A multi-layer perceptron (MLP) classifier was developed to predict infection status. CVB3-infected SOCS-1-transgenic (tg) mice were treated with immune- or non-immune sera on days 2 and 3 p.i., followed by monitoring of diabetes development. Results Frequent blood sampling and longitudinal measurement of the blood proteome revealed transient molecular changes in virus-infected animals that would have been missed with less frequent sampling. The MLP classifier predicted infection status after day 2 p.i. with over 90% accuracy. Treatment with immune sera on day 2 p.i. prevented diabetes development in all (100%) of CVB3-infected SOCS-1-tg NOD mice while five out of eight (62.5%) of the CVB3-infected controls treated with non-immune sera developed diabetes. Conclusions/interpretation Our study demonstrates the utility of frequently collected DBS samples to monitor dynamic proteome changes induced by an environmental trigger during the presymptomatic phase of type 1 diabetes. This approach enables disease interception and can be translated into human initiatives, offering a new method for early detection and intervention in type 1 diabetes. Data and code availability Additional data available at https://doi.org/10.17044/scilifelab.27368322 . Additional visualisations are presented in the Shiny app interface https://mouse-dbs-profiling.serve.scilifelab.se/ . Graphical Abstract

Aims/hypothesis Type 1 diabetes is characterised by the destruction of pancreatic beta cells. Genetic factors account for approximately 50% of the total risk, with variants in the HLA region contributing to half of this genetic risk. Research has historically focused on populations of European ancestry. We developed HLA-focused type 1 diabetes genetic risk scores (T1D GRSHLA) using SNPs or HLA alleles from four ancestry groups (admixed African [AFR; T1D GRSHLA-AFR], admixed American [AMR; T1D GRSHLA-AMR], European [EUR; T1D GRSHLA-EUR] and Finnish [FIN; T1D GRSHLA-FIN]). We also developed an across-ancestry GRS (ALL; T1D GRSHLA-ALL). We assessed the performance of the GRS in each population to determine the transferability of constructed scores. Methods A total of 41,689 samples and 13,695 SNPs in the HLA region were genotyped, with HLA alleles imputed using the HLA-TAPAS multi-ethnic reference panel. Conditionally independent SNPs and HLA alleles associated with type 1 diabetes were identified in each population group to construct T1D GRSHLA models. Generated T1D GRSHLA models were used to predict HLA-focused type 1 diabetes genetic risk across four ancestry groups. The performance of each T1D GRSHLA model was assessed using receiver operating characteristic (ROC) AUCs, and compared statistically. Results Each T1D GRSHLA model included a different number of conditionally independent HLA-region SNPs (AFR, n=5; AMR, n=3; EUR, n=38; FIN, n=6; ALL, n=36) and HLA alleles (AFR, n=6; AMR, n=5; EUR, n=40; FIN, n=8; ALL, n=41). The ROC AUC values for the T1D GRSHLA from SNPs or HLA alleles were similar, and ranged from 0.73 (T1D GRSHLA-allele-AMR applied to FIN) to 0.88 (T1D GRSHLA-allele-EUR applied to EUR). The ROC AUC using the combined set of conditionally independent SNPs (T1D GRSHLA-SNP-ALL) or HLA alleles (T1D GRSHLA-allele-ALL) performed uniformly well across all ancestry groups, with values ranging from 0.82 to 0.88 for SNPs and 0.80 to 0.87 for HLA alleles. Conclusions/interpretation T1D GRSHLA models derived from SNPs performed equivalently to those derived from HLA alleles across ancestries. In addition, T1D GRSHLA-SNP-ALL and GRSHLA-allele-ALL models had consistently high ROC AUC values when applied across ancestry groups. Larger studies in more diverse populations are needed to better assess the transferability of T1D GRSHLA across ancestries. Graphical Abstract

Aims/hypothesis Despite being commonly prescribed to treat painful diabetic peripheral neuropathy (DPN), the impact on the brain of long-term opioid use as analgesia is unknown. The aim of this study was to determine the structural and functional brain alterations associated with prescription opioid use in a large cohort of people with painful DPN. Methods A total of 82 patients with diabetes were enrolled: 57 with painful DPN (18 with long-term opioid prescription [O+ individuals] and 39 who were not prescribed opioids [O− individuals]) and a control group of 25 patients with diabetes but without DPN (no DPN) matched for age (± 2 years), sex and type of diabetes. All participants underwent detailed clinical/neurophysiological assessment and brain MRI at 3 T, and a subset (14 in each group, n=42) also underwent resting-state functional MRI. Results O+ individuals had greater caudate volume (ANOVA, p=0.03) compared with O− individuals (p=0.03) and those with no DPN (p=0.01). Functional connectivity was lower between the caudate and thalamus (r β = −0.24, seed-level correction −3.9, pFDR ≤0.05) in O+ individuals compared to those with no DPN. Moreover, seed-to-voxel analysis using caudate as the seed showed a significantly lower functional connectivity in O+ individuals compared with O− individuals in a cluster encompassing the superior frontal gyri bilaterally. Conclusions/interpretation We demonstrate that disruption of dopaminergic pathways occurs within the brain when opioids are used for analgesic purposes for painful DPN, which may reflect alterations in reward systems. This study has important clinical implications, as the measures of dopaminergic pathways found in this study may represent neuroimaging biomarkers that could be used to diagnose and monitor the negative consequences of prescription opioid use. Graphical Abstract

In economically developed countries most lean individuals presenting with insulin-dependent diabetes have autoimmune type 1 diabetes. However, in many rural areas of low- and middle-income countries (LMIC), 40–50% of individuals with a similar clinical presentation are negative for diabetes-associated autoantibodies at initial clinical presentation. The phenotype differs from the classical presentation of type 1 diabetes even in those with evidence of an autoimmune process: altered autoantibody profile; later peak age of onset; and, in those with post-pubertal clinical presentation, more marked male predominance. The incidence of insulin-dependent diabetes in LMIC is low, even when assessing those with and without autoantibodies together. A framework of possible pathophysiological mechanisms underlying the observed phenotypic differences is presented to explain how chronic undernutrition and micronutrient deficiencies might alter the presentation of insulin-dependent diabetes. Inhabitants of rural sub-Saharan Africa (SSA) depend almost entirely on staple foods grown locally in nutrient-deficient soil. The resulting chronic undernutrition, often intergenerational, affects linear growth and body morphology, and has direct immune and non-immune effects on beta cell development and function. Undernutrition directly affects thymic function, alters the autoimmune profile and is often associated with social deprivation and parasitic infection, both of which can delay and modify the (auto)immune response. Non-immune effects of undernutrition include beta cell stress, associated with apoptosis and formation of neoantigens. That environmental effects of undernutrition and social deprivation affect the altered insulin-dependent diabetes phenotype is shown by the movement back towards a classical type 1 diabetes phenotype in offspring of emigrants from SSA who are born in and develop insulin-dependent diabetes in an economically developed country. The degree of phenotype change depends on how long the parents have lived in their adopted country. It has recently been proposed that insulin-dependent diabetes in those who are mal/undernourished be called type 5 diabetes. There is need for clinician recognition of the altered phenotype(s) of insulin-dependent diabetes resulting from chronic undernutrition in rural LMIC. Additionally, changes in agricultural practice are needed to improve the nutrient content of food consumed by the rural population. Graphical Abstract

Recent advances in genome-wide approaches, the availability of isolated human islets for research and the evaluation of novel incretin mimetics in large clinical trials have brought about remarkable progress in our understanding of the role of the pancreatic beta cell in type 2 diabetes. Here, we review key developments in type 2 diabetes initiation, progression and remission, focusing mostly on human studies published in the last 5 years. Progress in multi-omics technologies has enabled researchers to identify links between type 2 diabetes risk variants and gene regulatory networks in islet endocrine cells that control beta cell development, function and stress resilience. These studies support the notion that early abnormalities in insulin secretion, rather than a reduction in beta cell mass, play a fundamental and primary role in early type 2 diabetes pathogenesis. Contributing to these intrinsic beta cell defects are various pathogenic signals from other (endocrine and non-endocrine) islet cells, the exocrine pancreas, the gut and insulin-sensitive tissues. It has also become apparent that beta cells comprise a heterogeneous population that responds differently to stress situations and that sex-related differences in beta cell responses should not be underestimated. Finally, human clinical trials have clearly demonstrated that diabetes remission can be achieved using glucose-lowering therapies and particularly strategies focused on weight loss, including bariatric surgery and, more recently, the use of highly efficient new drugs targeting the incretin system. While progress in the last 5 years has been significant, much remains to be uncovered to bring these advances to the clinic and thereby alleviate the dramatic consequences of type 2 diabetes complications for the hundreds of millions of people who live with this disease. Graphical Abstract

Aims/hypothesis Previous studies have indicated that 29-amino-acid glucagon (i.e. ‘pancreatic’ glucagon) circulates in totally pancreatectomised individuals and that a postprandial glucagon response can be detected. Using a glucagon receptor antagonist (GRA), we investigated the possible role of extrapancreatic glucagon on glucose, lipid and amino acid metabolism in totally pancreatectomised individuals. Method In a randomised, crossover study, nine totally pancreatectomised individuals and nine matched healthy control individuals were given, in randomised order (planned on the website www.random.org ), 300 mg GRA (LY2409021; Eli Lilly) or placebo 10 h before two 3 h OGTTs. The experiment was double-masked (i.e. both participants and investigator were masked for the type of the experimental day [day A vs day B]). The key inclusion criteria for the healthy control participants were age >18 years, normal fasting plasma glucose and HbA1c 31–44 mmol/mol (6.0–7.2%), haemoglobin >7.0 mmol/l (men) / >6.5 mmol/l (women) and informed consent. Key inclusion criteria for the pancreatectomised individuals were age >18 years, haemoglobin in the normal range and informed consent. The primary endpoint was the difference in plasma glucose excursions between study days. Results Glucagon concentrations remained unchanged from fasting concentrations during the OGTT in the totally pancreatectomised individuals on both study days and circulating glucose, lipids and amino acid levels were unaffected by treatment with LY2409021 compared with placebo. In the control group, LY2409021 resulted in relevant pharmacodynamic effects, including lower fasting plasma glucose (4.7 [0.1] vs 5.2 [0.1] mmol/l, p=0.001) and augmented concentrations of amino acids in plasma, compared with placebo. Conclusions/interpretation We conclude that inhibition of the glucagon receptor using LY2409021 during OGTT in totally pancreatectomised individuals does not produce detectable effects on glucose, lipid or amino acid metabolism, ruling out metabolic effects of extrapancreatic glucagon. Trial registration ClinicalTrials.gov (NCT02944110). Funding This study was supported by grants from the Aase and Ejnar Danielsen’s Foundation and the Novo Nordisk Foundation. Graphical Abstract

Aims/hypothesis The loss of pancreatic beta cell mass and identity is a hallmark of diabetes. While factors such as beta cell overwork (insulin hypersecretion) and elevated intracellular calcium have been implicated, beta cell identity loss also occurs in KATP gain-of-function (KATP-GOF) mice, a model of human neonatal diabetes, even in the absence of these factors. This suggests additional underlying mechanisms. Autophagy, a key process for cellular homeostasis, is impaired in the islets and beta cells of both type 1 and type 2 diabetes, but its role in monogenic diabetes with insulin secretory deficiency remains unclear. We hypothesise that autophagy dysfunction contributes to beta cell identity loss in KATP-GOF mice, and that intermittent fasting (IF) can restore autophagic flux, thereby preserving functional beta cell mass. Methods To test this, adult tamoxifen-inducible KATP-GOF mice and littermate controls were randomly assigned to two groups: (1) chow diet ad libitum; and (2) chow diet with alternate-day IF. Results KATP-GOF mice fed ad libitum developed severe hyperglycaemia due to impaired insulin secretion. This was followed by a reduction in insulin content, disruption of beta cell autophagic flux, autophagosome accumulation and, ultimately, the loss of beta cell identity and dedifferentiation. In contrast, KATP-GOF mice subjected to alternate-day IF exhibited lower blood glucose levels, improved mitochondrial morphology, restoration of autophagic flux and reestablishment of beta cell identity. Conclusions/interpretation This study provides the first evidence of autophagy impairment in non-obese, insulin secretory-deficient, KATP-induced diabetes mice and demonstrates that IF restores both autophagic flux and beta cell identity. This finding suggests that similar mechanisms may contribute to beta cell dysfunction in other forms of diabetes. Graphical Abstract

Aims/hypothesis The incidence of type 1 diabetes increased during the pandemic in various countries. SARS-CoV-2 infections may trigger the development of type 1 diabetes, but the evidence is inconclusive. This study aimed to assess trends in type 1 diabetes incidence between 2007 and 2023, and to quantify the association between SARS-CoV-2 infections and the risk for developing type 1 diabetes. Methods The study included all individuals under 30 years old registered in Sweden. Deviations in type 1 diabetes incidence from pre-pandemic trends (2007–2019) were assessed for each pandemic year (2020–2023) using Poisson regression. The effect of SARS-CoV-2 infections was assessed using Cox proportional hazards models in a cohort of infected individuals with five control individuals from the infection date of the case, matched by birth year, sex and region. Results Compared with the predicted linear trend, type 1 diabetes incidence increased by 12% during 2021 (incidence rate ratio [IRR] 1.12; 95% CI 1.06, 1.19) and 9% during 2022 (IRR 1.09; 95% CI 1.02, 1.16), but reverted to pre-pandemic trends in 2023. Overall, the adjusted HR for developing type 1 diabetes after SARS-CoV-2 infection was 0.96 (95% CI 0.79, 1.16). Children between 5 and 10 years old were more likely to develop type 1 diabetes within the first 28 days after infection (HR 2.68; 95% CI 1.22, 5.89), although their hazard over the whole follow-up period was not increased. Conclusions/interpretation Sweden, with its non-restrictive pandemic response, saw a transient increase in type 1 diabetes incidence that was only partially associated with SARS-CoV-2 infections. Other explanations should be investigated, including environmental and lifestyle factors. Graphical Abstract

Aims/hypothesis The role of intra-pancreatic lipid and collagen in type 2 diabetes pathogenesis remains unclear. We sought to examine this in pancreases from organ donors with and without diabetes. Methods Tissue biopsies from 36 adult donor pancreases with/without type 2 diabetes were collected from 16 anatomically defined regions, with H&E, Sirius Red Fast Green and chromogranin A immunohistochemical staining and quantification performed. Intracellular lipid droplet area was quantified using transmission electron microscopy in acinar, islet endocrine, beta and alpha cells identified through ultrastructural morphology. Results Increasing adipocyte proportional area was associated with increasing pancreas donor BMI (r=0.385, p=0.021), decreased acinar area (r=−0.762, p<0.001) and increased endocrine mass (r=0.749, p<0.001). Fibrosis was not associated with BMI, acinar area or endocrine mass. Type 2 diabetes was associated with decreased islet circularity and reduced beta:alpha cell ratio but endocrine mass was not affected. Adipocyte and fibrosis proportional areas were highest in donors with diabetes but not associated with each other. Pancreases with high fat and those with high fibrosis (>40% proportional area) appeared to form two separate subgroups. All donors with insulin-treated diabetes had a high collagen proportional area. Fibrosis but not adipocytosis was associated with decreased beta:alpha cell ratio. There was an inverse relationship between pancreatic adipocytosis and intra-acinar cell lipid content (r=−0.490, p=0.003), with the lowest levels seen in type 2 diabetes. Beta cell lipid content was associated with BMI but not type 2 diabetes. Conclusions/interpretation Systematic human pancreatic analysis revealed two separate type 2 diabetes phenotypes: fatty, associated with central obesity; and fibrotic, associated with reduced beta cell mass without central obesity. This suggests distinct underlying pathogenic mechanisms and has potential for developing personalised disease-modifying therapeutics. Graphical Abstract

Aims/hypothesis Faecal microbiota transplantation (FMT) may alleviate gastrointestinal symptoms in individuals with diabetic gastroenteropathy, as demonstrated in a recent placebo-controlled trial. In most participants, symptom relief was transient, raising the need for repeated treatments. This study assessed the long-term efficacy, safety and feasibility of repeated, on-demand FMT as a maintenance treatment in this patient population. Methods All 20 participants from the randomised clinical trial were offered extended open-label treatment with FMT. Symptom assessments were conducted by telephone every 2–3 months using the Gastrointestinal Symptom Rating Scale for Irritable Bowel Syndrome (GSRS-IBS). Secondary measures included bowel movement frequency, stool consistency assessed using the Bristol Stool Scale, perceived treatment benefit on a seven-point Likert scale, and adverse events (AEs). FMT was primarily given as oral capsules, and colonoscopy was used for participants who could not swallow capsules. Results Of the original 20 participants, 17 were included in the present study and followed from September 2021 to December 2024, with a median Duration of follow-up of 33.2 months (range 14.7–39.1 months). Participants received a total of 95 FMT treatments, with a median of five per participant and a median interval of 5.3 months between treatments. FMT induced consistent symptom relief, with reduced GSRS-IBS scores across multiple treatments. At the last FMT treatment provided, the mean GSRS-IBS score had decreased from 60 (95% CI 54, 66) at baseline to 35 (95% CI 29, 40), with a mean difference of −25 (95% CI −18, −33). The occurrence of frequent bowel movements 2 weeks after treatment (> 7 per day) decreased from 19% (95% CI 10%, 28%) to 3% (95% CI 0%, 7%). Stool consistency improved after treatment, and the frequency of normal stool types (Bristol Stool Scale score 3–5) increased from 28% (95% CI 18%, 39%) to 76% (95% CI 66%, 86%). Participant satisfaction was high, with 86% reporting considerable benefits (Likert scores 5–7). Repeated FMT was generally well tolerated, with most AEs being mild and self-limiting. Fifteen serious AEs were documented, of which only one was deemed to be possibly related to FMT. Conclusions/interpretation Repeated, on-demand FMT is effective and safe for long-term treatment of individuals with type 1 diabetes and severe diabetic gastroenteropathy. Trial registration ClinicalTrials.gov NCT04749030 Funding The study was funded by a Steno Collaborative Grant (no. 0058906) Graphical Abstract