Type 1 diabetes (T1D) results from autoimmune-mediated destruction of insulin-producing β cells in the pancreatic islet. This process is modulated by pro-inflammatory cytokine signaling, which has been previously shown to alter protein expression in ex vivo islets. Herein, we applied top-down proteomics to globally evaluate proteoforms from human islets treated with proinflammatory cytokines (interferon-γ and interleukin-1β). We measured 1636 unique proteoforms across six donors and two time points (control and 24 h post-treatment) and observed consistent changes in abundance across the glicentin-related pancreatic polypeptide (GRPP) and major proglucagon fragment regions of glucagon, as well as the LF-19/catestatin and vasostatin-1/2 region of chromogranin-A. We also observe several proteoforms that increase after cytokine-treatment or are exclusively observed after cytokine-treatment, including forms of beta-2 microglobulin (B2M), high-mobility group N2 protein (HMGN2), and chemokine (C-X-C motif) ligands (CXCL). Together, our quantitative results provide a baseline proteoform profile for human islets and identify several proteoforms that may serve as interesting candidate markers for T1D progression or therapeutic intervention. SUMMARY: This work applies a top-down proteomics workflow for the characterization and label-free quantification of proteoforms from human islets in the context of inflammation. The workflow is optimized for challenges unique to the islet proteome including high disulfide-linkage content and frequent truncation events, resulting in many proteoforms < 5kDa. There are limited examples of top-down proteomics characterization of human islets, thus this study provides a baseline characterization of the proteoforms of major hormones including chromogranin-A (CHGA), chromogranin-B/ secretogranin-1 (CHGB/SCG1), chromogranin-C/ secretogranin-2 (CHGC/SCG2), islet amyloid polypeptide (amylin/IAPP), insulin (INS), glucagon (GCG), pancreatic polypeptide prohormone (PPY), somatostatin (SST), and neurosecretory protein VGF (VGF). The quantitative results of proteoform abundances before and after cytokine treatment, which mimics the proinflammatory environment during T1D progression, provides interesting insights on how prohormone processing is altered under a proinflammatory environment.

N⁶-methyladenosine (m⁶A) is among the most abundant mRNA modifications, yet its cell-type-specific regulatory roles remain unclear. Here we show that m⁶A methyltransferase-like 14 (METTL14) differentially regulates transcriptome in brown versus white adipose tissue (BAT and WAT), leading to divergent metabolic outcomes. In humans and mice with insulin resistance, METTL14 expression differs significantly from BAT and WAT in the context of its correlation with insulin sensitivity. Mettl14-knockout in BAT promotes prostaglandin secretion, improving systemic insulin sensitivity. Conversely, Mettl14-knockout in WAT triggers adipocyte apoptosis and systemic insulin resistance. m⁶A-seq and RNA-seq integration revealed upregulated prostaglandin biosynthesis pathways in BAT and apoptotic pathways in WAT with Mettl14 deficiency. Stable METTL14-knockout hBAs/hWAs show METTL14-mediated m⁶A promotes mRNA decay of PTGES2 and CBR1 in hBAs and TRAIL and TNFR1 in hWAs. These data shed light on the ability of m⁶A to impact metabolism in a cell-type-specific manner with implications for influencing the pathophysiology of metabolic diseases.

© 2025. The Author(s).

Aims/hypothesis: Progression to type 1 diabetes is associated with genetic factors, the presence of autoantibodies and a decline in beta cell insulin secretion in response to glucose. Very little is known regarding the molecular changes that occur in human insulin-secreting beta cells prior to the onset of type 1 diabetes. Herein, we applied an unbiased proteomics approach to identify changes in proteins and potential mechanisms of islet dysfunction in islet-autoantibody-positive organ donors with pre-symptomatic stage 1 type 1 diabetes (HbA_1c ≤42 mmol/mol [6.0%]). We aimed to identify pathways in islets that are indicative of beta cell dysfunction.

Methods: Multiple islet sections were collected through laser microdissection of frozen pancreatic tissues from organ donors positive for single or multiple islet autoantibodies (AAb⁺, n=5), and age (±2 years)- and sex-matched non-diabetic (ND) control donors ( n=5) obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD). Islet sections were subjected to MS-based proteomics and analysed with label-free quantification followed by pathway and functional annotations.

Results: Analyses resulted in ~4500 proteins identified with low false discovery rate (<1%), with 2165 proteins reliably quantified in every islet sample. We observed large inter-donor variations that presented a challenge for statistical analysis of proteome changes between donor groups. We therefore focused on only the donors with stage 1 type 1 diabetes who were positive for multiple autoantibodies (mAAb⁺, n=3) and genetic risk compared with their matched ND controls (n=3) for the final statistical analysis. Approximately 10% of the proteins (n=202) were significantly different (unadjusted p<0.025, q<0.15) for mAAb⁺ vs ND donor islets. The significant alterations clustered around major functions for upregulation in the immune response and glycolysis, and downregulation in endoplasmic reticulum (ER) stress response as well as protein translation and synthesis. The observed proteome changes were further supported by several independent published datasets, including a proteomics dataset from in vitro proinflammatory cytokine-treated human islets and single-cell RNA-seq datasets from AAb⁺ individuals.

Conclusions/interpretation: In situ human islet proteome alterations in stage 1 type 1 diabetes centred around several major functional categories, including an expected increase in immune response genes (elevated antigen presentation/HLA), with decreases in protein synthesis and ER stress response, as well as compensatory metabolic response. The dataset serves as a proteomics resource for future studies on beta cell changes during type 1 diabetes progression and pathogenesis.

Background
Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) is a ubiquitously expressed mitochondrial enzyme with a role in the degradation of fatty acids. Because the protein also is a negative regulator of insulin secretion in pancreatic β-cells, inactivating mutations in the SCHAD gene (HADH) cause congenital hyperinsulinism of infancy (CHI) and severe hypoglycemia. Here we sought to identify novel interaction partners of SCHAD that might be particularly relevant for the endocrine pancreas.

Results
Employing the SCHAD protein as bait, we performed yeast 2-hybrid screening of a cDNA library made from human islets of Langerhans. Surprisingly, the screening revealed the intermediate filament protein keratin 8 (K8) as a putative interaction partner of SCHAD with very high confidence. Previous reports have linked K8 to glucose homeostasis, and we confirmed the SCHAD interaction by co-immunoprecipitation in HEK293 cells. SCHAD and K8 expression were then characterized in the human β-cell model EndoC-βH1. By using proximity ligation assay, we demonstrated that stimulating the cells with a high level of glucose triggered a transient increase in the interaction. However, when studying knockout mice, we found that the loss of either K8 or SCHAD did not change the expression level of the other interaction partner. Still, when K8 knockout mice were challenged with a ketogenic diet, upregulation of SCHAD expression was blunted compared to the upregulation observed in wildtype littermates.

Conclusions
We propose that the SCHAD protein interacts with K8 in a way that might be relevant for proper functioning of the pancreatic β-cell. Whether the SCHAD-K8 interaction influences the phenotype of CHI remains to be demonstrated.

The role of N6-methyladenosine (m6A) RNA methylation in liver regeneration is unclear. This study aimed to determine the role of m6A methylation in liver regeneration after a 70% hepatectomy (HEPA) using liver-specific methyltransferase-like 14 (Mettl14) knockout (KO) male mice. Analysis was conducted on postoperative days 1, 3, or 7 (HEPA1, 3, or 7) in control (Flox) mice. In Flox mice, cyclin D1 protein expression was highest on postoperative day 3 (HEPA3) consistent with a dynamic increase in hepatocyte replication. The abundance of Mettl14 protein presented a similar pattern on HEPA3. Then, we performed hepatectomy in Mettl14 KOs (M14KO) and Flox controls and observed significantly higher postsurgical mortality in mutants. In Flox mice, cyclin D1 protein levels and Ki-67 were markedly increased on HEPA3 compared to sham operation, while being downregulated in M14KO. Characterizing the m6A epitranscriptomic changes in Flox mice after hepatectomy and contrasting them to hepatectomy in M14KO in HEPA3 revealed enrichment for gene ontology terms associated with endoplasmic reticulum, inflammation, and apoptosis. Differentially methylated genes in M14KO compared to Flox on HEPA3 were also enriched for peroxisome proliferator-activated receptor (PPAR) and AMPK signaling. Finally, we identified hypomethylated transcripts involved in fibrinogen synthesis, such as Fga, Fgb, and Fgg, by comparing differentially m6A-decorated genes in M14KO vs. Flox on HEPA3. Knockdown of fibrinogen leads to suppression of proliferation via activation of p21 protein in AML12 cells. Together, these data point to m6A RNA methylation being significant in decorating genes involved in fibrinogen synthesis in liver regeneration.

Glucagon, secreted by pancreatic alpha cells, is essential for maintaining normal blood glucose levels. In type 1 and advanced type 2 diabetes, alpha cells often fail to respond to low glucose, yet the mechanisms underlying  their stress resistance remain unclear. To investigate this, we performed a genome-wide CRISPR screen and identify Sec31A, a gene involved in transporting proteins from the endoplasmic reticulum (ER), as a key regulator of alpha cell survival under stress. We show that loss of Sec31A enhances survival in stressed mouse alpha cells and in C. elegans. In human islets, SEC31A expression increases in alpha cells under inflammatory stress, and this upregulation is reversed by reducing ER stress. Functional studies in lab-grown human islet clusters reveal distinct responses in alpha versus beta cells following Sec31A suppression. We also find that Sec31A interacts with the insulin receptor, suggesting a link between stress adaptation and insulin signaling in alpha cells.

Insulin-like growth factor-2 receptor (IGF2R), also known as cation-independent mannose-6-phosphate receptor, is localized in cytosolic vesicles and is unique in its ability to transport enzymes to the lysosome and to clear IGF2 from the cell surface by acting as a scavenger receptor. To evaluate the direct role of IGF2R in β-cell biology, we undertook complementary in vitro knockdown and in vivo knockout approaches. A β-cell line with a stable knockdown of IGF2R (IGF2RKD) exhibited decreased glucose-induced insulin secretion and enhanced cell proliferation. Tamoxifen-inducible β-cell–specific IGF2R knockout mice exhibited impaired glucose tolerance and blunted insulin secretion after high-fat-diet loading that was likely secondary to reduced β-cell mass due to attenuated proliferation. β-cells with IGF2RKD had fewer autophagosomes after starvation and reduced expression of p62, LC3B, and ULK1. Aged mice also had impaired autophagy in βIGF2R-deficient β-cells. Reduced IGF2R function and N6-methyladenosine (m6A) mRNA methylation were observed in islets from both mouse and human type 2 diabetes. Taken together, these data point to IGF2R as an important regulator of insulin secretion, cell proliferation, and autophagy in mammalian β-cells.