Pancreatic β-cells adapt to increased insulin demand through coordinated functional and molecular responses. However, in diabetes, these adaptive pathways eventually fail, driving β-cell dysfunction and demise. One of these pathways is unfolded protein response (UPR), a complex network of sensors and effectors that cooperate to restore endoplasmic reticulum (ER) function under stress. To define the early UPR programs engaged during β-cell adaptation, we employed a murine model of partial β-cell ablation that preserves normoglycemia while imposing an increased secretory workload on the remaining β-cells. Integrating bulk islet transcriptomics with imaging, ultrastructural and physiological analyses, we identified dynamic molecular and functional adaptations during the compensatory phase. The initial transcriptional response was characterised by induction of apoptosis genes and a stress response. This was later replaced by activation of adaptive UPR pathways accompanied by increased insulin processing. Nuclear Atf4 and Xbp1 increased transiently, consistent with engagement of multiple UPR branches through different mechanisms to restore homeostasis. Functionally, partially ablated islets displayed enhanced secretory competence during an acute secondary ER stress challenge, although this adaptive advantage was lost during prolonged stress. Together, these findings define a transient adaptive UPR program that sustains β-cell function during the early response to β-cell loss and provide insight into mechanisms that may be leveraged to enhance β-cell resilience in diabetes.

Regulatory circuits driving regional cell fate specification and lineage restriction decisions are not fully understood. The molecular mechanisms by which Hedgehog signaling controls lineage segregation in the posterior foregut remain unclear. Here, we employed bulk and single-cell transcriptomics, microscopy, physiology, and genetic cell tracing in differentiating human induced pluripotent stem cells and mouse transgenic models to uncover an essential autoregulatory loop between Hnf1a and Hedgehog signaling. Hnf1a abrogation initiates a domino effect leading to a drift in foregut cell specification toward duodenal cell identity instead of pancreatic fate. This was replicated in vivo in mice. We show that a common dominant negative Hnf1a mutation disrupts GLI3 processing by cilium proteins in the posterior foregut, thus impeding its own upregulation in response to Hedgehog signaling inhibition. In the context of this defective loop, we identified a network of deregulated selector genes responsible for the lineage segregation changes. These may be relevant for changes in the liver, pancreas, and gut seen in patients with mutations in HNF1A.

This discovery study investigated the impact of MODY-associated mutations in hepatocyte nuclear factors HNF1A and HNF1B on the cellular proteome, aiming to identify affected pathways and advance understanding of diabetes pathogenesis. Human induced pluripotent stem cells (hiPSCs) carrying the HNF1A frameshift mutation (p.Pro291fsinsC) were differentiated into pancreatic progenitors, and renal proximal tubule epithelial cells (RPTECs) were engineered to overexpress HNF1B with the S148L point mutation. Label-free quantitative proteomics was performed using data-dependent and data-independent acquisition on Orbitrap and timsTOF mass spectrometers. Pathway enrichment was analyzed using Qiagen IPA, Hallmark gene sets, and STRING networks. Comprehensive proteome coverage (over 7000 proteins) revealed consistent downregulation of oxidative phosphorylation, mitochondrial function, and interferon signaling pathways. Both models exhibited suppression of innate immune responses, with overlapping downregulated proteins, including members of the OAS, IFIT, and MX1 families. Using label-free proteomics, we show that MODY-associated mutations in HNF1A and HNF1B suppress mitochondrial function and interferon signaling and are additionally associated with reduced abundance of predicted targets such as A1CF as well as diabetes-related proteins including SCGN, supporting their role in diabetes pathogenesis.

Introduction Neuroendocrine neoplasms represent a rare and poorly understood collection of malignancies. A better understanding of their biology is needed to improve treatment options. Male predominance to the incidence of neuroendocrine neoplasms of the thymus suggests a biological basis for this observation. Methods This single-institution, retrospective cohort study evaluated androgen receptor (AR) expression and other clinicopathologic features in thymic neuroendocrine neoplasms compared with thymic epithelial tumors and neuroendocrine neoplasms of other sites. Expression of neuroendocrine and prostate markers was also assessed. For further molecular characterization, copy number analysis of AR and other relevant genes was assessed by single nucleotide polymorphism array. Last, analysis of single genes and thymic epithelial gene sets was performed using published thymic tumor transcriptomes. Results A cohort was assembled from 17 patients with neuroendocrine malignancies of primary thymic origin with assessable tissue for immunohistochemical analysis. The cohort was predominantly male (12 males, five females). Immunohistochemical analysis of the AR demonstrated positive staining in nine cases, all of which were male. Compared with thymic neuroendocrine neoplasms, AR expression is considerably less frequent in thymomas, thymic carcinoma, and neuroendocrine neoplasms of other sites of origin. No relevant copy number changes in the AR were identified. Further molecular characterization revealed expression of ASCL1 and limited expression of common prostate lineage and differentiation markers within our cohort, including thymic neuroendocrine epithelial gene set enrichment in published transcriptomes of thymic neuroendocrine neoplasms. Conclusion The AR is expressed in a considerable fraction of thymic neuroendocrine neoplasms and is associated with male predominance. This observation has implications for investigation of androgen deprivation and receptor blockade.

Quantification of subcellular structures such as nuclei and cytoplasmic proteins using staining methods based on fluorescent dyes or fluorescently tagged antibodies are widely used in scientific research. Accurate high-throughput quantitation of these assays can be time consuming and challenging. Here, we present our FIJI based Semi-Automated counting Macro termed SAM, and we validate its accuracy against manual counting and other automated counting methods. By introducing this automated quantification tool, we aim to contribute to the ongoing efforts to enhance the reliability, efficiency, and standardization of immunostaining analysis in the field of diabetes research and beyond.

We investigated the proteomic consequences of MODY-associated mutations in the transcription factors HNF1A and HNF1B using two cell line models. Quantitative label-free mass spectrometry, employing both data-dependent and data-independent acquisition methods, revealed consistent suppression of energy metabolism and interferon signaling pathways. Pathway and protein interaction analyses confirmed these findings. In cells with the HNF1A frameshift mutation, several predicted transcriptional targets, including A1CF , were significantly downregulated. These results reinforce the link between HNF1A/HNF1B function and mitochondrial activity as well as innate immune signaling, providing a foundation for further mechanistic studies and causal inference research.

Background In the past decades tissue/cell targeted single gene modifications using transgenic systems became a main-stream practice aimed at demultiplexing tissue- and cell-specific gene function. Yet, targeting of many genes and cell types can cause systemic effects, impacting the functionality of other off-target organs. This can further generate a discrete dysfunction loop fueling back to the targeted cell altering their profile readout, effect demultiplexing and results interpretation. Despite the high impact of such scenario especially in the study of endocrine organs, most research is focused on targeted mutation-bearing cell population, while the other organs bearing intact candidate gene activity, receive no or limited attention. Results To assess the potential readout bias caused by off-target organs, we performed here a focused pilot transcriptomics study to map the effects on liver of a monogenic diabetes gene mutation restricted to insulin-expressing beta-cells. Mice with beta-cell restricted disfunction were mildly hyperglycemic and presented normal target gene levels in the liver. Despite normal expression, pathway analyses identified profound transcriptional prolife changes in the liver. These involved the dysregulation of lipid metabolism and extracellular matrix organization, cholesterol biosynthesis being further exacerbated by HFD, consistent with a systemic factor effect such as chronically elevated blood sugar levels. Furthermore, key markers of hepatic steatosis were highly increased, with the livers’ histopathology reflecting lipid droplet accumulation. As hepatic steatosis is an important cause of hepatic insulin resistance that can further alter beta-cell function, the interpretation of the transcriptional background in the targeted beta-cell population must be performed with care. Conclusions Based on this pilot we conclude that multi-organ dysfunction loops can drastically change the read-out in the mutated cell complicating the effect separation. Thus investigating off-target organs is crucial, especially when characterizing genes and cell populations involved in endocrine regulation.

AIM:HNF1A-MODY, the most prevalent form of monogenic diabetes, displays incomplete penetrance, indicating the involvement of other environmental and genetic factors in the disease etiology. Currently, it is largely unknown what the influence of environmental factors, such as toxins or diet, is on HNF1A-MODY onset and progression. Here we address this issue by exploring the impact of diet on islet and insulin-secreting beta-cells in the context of HNF1A mutation. METHODS:Transgenic mice allowing the specific Hnf1a mutation in insulin-secreting beta-cells were exposed to four distinct dietary regimens including combinations of high-fat diet and caloric restriction. In vitro stem cell islets bearing the HNF1AP291fsinsC heterozygous mutation and their isogenic controls were used for validation in humans. The readouts included physiological tests, immunofluorescence, proteomics, bulk, and single-cell transcriptomics. RESULTS:Hnf1a-deficient beta-cells exhibited high sensitivity to dietary cues. Exposure to a high-fat diet exacerbated the glucose regulation defects, while caloric restriction significantly improved blood glucose levels in vivo, without perturbing islet architecture. The high-throughput methods identified changes in the Hnf1a-deficient beta-cells proteome landscape, involving conserved critical regulators of metabolic and growth processes, such as the Carbohydrate Response Element Binding Protein (Chrebp/Mlxipl) and ATP citrate lyase (Acly) among others. CONCLUSIONS:This study hallmarks the important impact of diet on Hnf1a-deficient beta-cells, stemming new therapeutic perspectives, such as future diet management approaches.

Differentiation of human induced pluripotent stem cells towards pancreatic islet endocrine cells is a complex process, involving the stepwise modulation of key developmental pathways, such as the Hedgehog signaling inhibition during early differentiation stages. In tandem with this active inhibition, key transcription factors for the islet endocrine cell fate, such as HNF1A, show specific changes in their expression patterns. Here we designed a pilot study aimed at investigating the potential interconnection between HH-signaling inhibition and the increase in the HNF1A expression during early regeneration, by inducing changes in the GLI code. This unveiled a link between the two, where GLI3-R mediated Hedgehog target genes inhibition is apparently required for HNF1A efficient expression.

The generation of insulin-producing cells from human-induced pluripotent stem cells holds great potential for diabetes modeling and treatment. However, existing protocols typically involve incubating cells with un-physiologically high concentrations of glucose, which often fail to generate fully functional IPCs. Here, we investigated the influence of high (20 mM) versus low (5.5 mM) glucose concentrations on IPCs differentiation in three hiPSC lines. In two hiPSC lines that were unable to differentiate to IPCs sufficiently, we found that high glucose during differentiation leads to a shortage of NKX6.1+ cells that have co-expression with PDX1 due to insufficient NKX6.1 gene activation, thus further reducing differentiation efficiency. Furthermore, high glucose during differentiation weakened mitochondrial respiration ability. In the third iPSC line, which is IPC differentiation amenable, glucose concentrations did not affect the PDX1/NKX6.1 expression and differentiation efficiency. In addition, glucose-stimulated insulin secretion was only seen in the differentiation under a high glucose condition. These IPCs have higher KATP channel activity and were linked to sufficient ABCC8 gene expression under a high glucose condition. These data suggest high glucose concentration during IPC differentiation is necessary to generate functional IPCs. However, in cell lines that were IPC differentiation unamenable, high glucose could worsen the situation.

Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.

Induced pluripotent stem cells as a source for generating pancreatic islet endocrine cells represent a great research tool for deciphering the molecular mechanisms of lineage commitment, a layered multi-step process. Additionally, targeted gene silencing by using GapmeRs, short antisense oligonucleotides, proved instrumental in studying the role of different developmental genes. Here we describe our approach to induce mTOR silencing by using specific GapmeRs during the differentiation of induced pluripotent stem cells toward pancreatic progenitors. We will describe our current differentiation protocol, the transfection procedure, and the quality control steps required for a successful experiment.

Besides its beneficial effect on weight loss, gastric bypass surgery (GBS) may impact the circulating levels of phospho- and sphingolipids. However, long-term effects have not been explored. To investigate alterations in lipidomic signatures associated with massive weight loss following GBS, we conducted direct infusion tandem mass spectrometry on serum and subcutaneous adipose tissue (SAT) samples collected in a longitudinal cohort of morbid obese patients prior to GBS and 1 year following the surgery. A tissue-specific rearrangement of 13% among over 400 phospholipid and sphingolipid species quantified in serum and SAT was observed 1 year following GBS, with a substantial reduction of ceramide levels and increased amount of hexosylceramides detected in both tissues. The comparison of these new lipidomic profiles with the serum and SAT lipidomes established from an independent cohort of lean and morbid obese subjects revealed that GBS partly restored the lipid alterations associated with morbid obesity.

Amid the advances in genomics, the availability of large reference panels of human haplotypes is key to account for human diversity within and across populations. However, mass spectrometry-based proteomics does not benefit from this information. To address this gap, we introduce ProHap, a Python-based tool that constructs protein sequence databases from phased genotypes of reference panels. ProHap enables researchers to account for haplotype diversity in proteomic searches.

Regeneration, the ability to replace injured tissues and organs, is a phenomenon commonly associated with lower vertebrates but is also observed in mammals, in specific tissues. In this study, we investigated the regenerative potential of pancreatic islets following moderate beta-cell loss in mice. Using a rapid model of moderate ablation, we observed a compensatory response characterized by transient inflammation and proliferation signatures, ultimately leading to the recovery of beta-cell identity and function. Interestingly, this proliferative response occurred independently of inflammation, as demonstrated in ablated immunodeficient mice. Furthermore, exposure to high-fat diet stimulated beta-cell proliferation but negatively impacted beta-cell function. In contrast, an equivalent slower ablation model revealed a delayed but similar proliferative response, suggesting proliferation as a common regenerative response. However, high-fat diet failed to promote proliferation in this model, indicating a differential response to metabolic stressors. Overall, our findings shed light on the complex interplay between beta-cell loss, inflammation, and stress in modulating pancreatic islet regeneration. Understanding these mechanisms could pave the way for novel therapeutic strategies based on beta-cell proliferation. Rapid and moderate 50% DT-induced beta-cell ablation prompts a compensatory response in the pancreatic islet, despite the absence of an obvious metabolic pressure to regenerate.

Animal longevity is a function of global vital organ functionality and, consequently, a complex polygenic trait. Yet, monogenic regulators controlling overall or organ-specific ageing exist, owing their conservation to their function in growth and development. Here, by using pathway analysis combined with wet-biology methods on several dynamic timelines, we identified Hnf1a as a novel master regulator of the maturation and ageing in the adult pancreatic islet during the first year of life. Conditional transgenic mice bearing suboptimal levels of this transcription factor in the pancreatic islets displayed age-dependent changes, with a profile echoing precocious maturation. Additionally, the comparative pathway analysis revealed a link between Hnf1a age-dependent regulation and immune signaling, which was confirmed in the ageing timeline of an overly immunodeficient mouse model. Last, the global proteome analysis of human islets spanning three decades of life largely backed the age-specific regulation observed in mice. Collectively, our results suggest a novel role of Hnf1a as a monogenic regulator of the maturation and ageing process in the pancreatic islet via a direct or indirect regulatory loop with immune signaling.