Biology subjects

Arrojo e Drigo, R.

Publications and source records attributed to Arrojo e Drigo, R..

13 recordsLinked to original sources

bioRxiv2026

MAFA drives pancreatic islet maturation through coordination of cell growth, metabolic maturation, and resilience to stress.

Stem cell-derived islets (SC-islets) are a promising source for replacing the loss of insulin-secreting {beta} cells in insulin-dependent diabetes patients, but compared to their primary human counter parts, SC-islets are considered functionally immature and do not immediately regulate blood glucose after transplantation. To better understand the molecular underpinnings of {beta} cell maturation, we genetically engineered human pluripotent stem cells (hPSCs) to control the induction of the {beta} cell-maturing transcription factor MAFA or a MAFA S64F mutant variant associated with premature {beta} cell aging and senescence in SC-islets. We employ a combination of in vitro assays, multi-omics analyses, and murine in vivo transplantation studies to demonstrate that MAFA remodeling of the SC-islet transcriptional landscape promotes SC-islet biogenesis and metabolic maturation giving rise to greater SC-islet resilience and insulin secretory capacity. Parallel MAFA S64F studies highlight the importance for compensatory mechanisms for sustaining accelerated oxidative metabolism and enhanced insulin secretion without inducing intracellular stress responses and islet dysfunction. Overall, these results reveal MAFA-governed transcriptional changes that drive SC-islet functional maturation, which provides the foundation for maturing SC-islets in vitro, preserving {beta} cell integrity, and improving SC-islet cell replacement therapies.

cell biology↗

bioRxiv2026

Correlative MIMS-EM imaging reveals metabolic turnover from organelle to organismal scales in C. elegans during dietary restriction

Metabolism is spatially compartmentalized across scales, from distinct tissues to cells and orga-nelles. However, most approaches for studying metabolic activity obscure spatial organization and intra-compartment heterogeneity within bulk biochemical measurements. On the other hand, multi-isotope mass spectrometry coupled with scanning electron microscopy (MIMS-EM) maps the fates of labeled nutrients in situ at nanometer-scale resolution, preserving ultrastructural con-text. Here we adapt MIMS-EM for Caenorhabditis elegans, where the compact metazoan body plan uniquely enables visualization of virtually all tissue types and their resident organelles within a single cross-sectional image. Using pulse-chase labeling of dietary carbon and nitrogen, we apply this approach to understanding the metabolic program induced in early stages of dietary restriction (DR). While DR is widely proposed to enhance organismal healthspan by enhancing broadscale turnover, proteomic studies have suggested more nuanced models. MIMS-EM across intact animals reveals that DR induces non-uniform effects between tissues and car-bon/nitrogen resources, accelerating carbon turnover in the muscle and hypodermis, but not in-testine. At the organelle scale, MIMS-EM revealed heterogeneity within mitochondrial networks that was independent of diet and stable over time. Spatial analysis of isotope signatures within intestinal mitochondrial networks also indicated greater similarity between neighboring mitochon-dria than distal mitochondria, supporting models of local mitochondrial mixing. Collectively, these results reveal that DR induces compartment- and resource-specific remodeling strategies across an intact animal while establishing C. elegans MIMS-EM as a powerful platform for multi-scale, integrative models of nutrient handling.

physiology↗

bioRxiv2026

QuantEM: An optimized platform of vision transformer-based models for segmentation and analysis of electron microscopy data

Electron microscopy (EM) is essential for resolving cellular ultrastructure, yet quantitative analysis remains limited by labor-intensive segmentation and the scarcity of generalizable models. Here we present QuantEM, an open-source platform for segmentation and analysis of EM data across imaging modalities, tissues, and species. We assembled the largest curated collection of intracellular EM datasets to date, comprising over 15,000 two-dimensional images and 1,700 three-dimensional acquisitions from more than 600 datasets, including nearly 4,000 newly released acquisitions. Using this resource, we trained an EM-specific vision transformer foundation model and systematically optimized adaptation strategies for organelle segmentation. QuantEM provides pretrained models for mitochondria, endoplasmic reticulum, nuclei, and lipid droplets, integrated with interactive proofreading and downstream quantitative analyses through standalone and napari interfaces. Across diverse naive datasets, QuantEM consistently matches or exceeds existing models on zero-shot segmentation while requiring less data for finetuning. We further demonstrate its utility by revealing previously unrecognized subcellular compartmentation of hepatic glucokinase using immuno-electron microscopy.

cell biology↗

bioRxiv2026

Calorie Restriction Up-regulates Islet PD-L1 Signaling and Decreases the Risk of Auto-immune Diabetes Onset in NOD Mice.

Type 1 diabetes (T1D) is an autoimmune disease where beta cells are destroyed by cytotoxic T cells. Calorie restriction (CR) enhances glucose homeostasis and promotes beta cell longevity and was used as therapeutic strategy for T1D prior to the discovery of insulin. However, a significant knowledge gap remains regarding its effects on beta cells during the pathogenesis of autoimmunity. We demonstrate that CR enhances glucose homeostasis, reduces beta cell load, and delays T1D onset in NOD mice. CR induced a largely post-mitotic beta cell state marked by selective loss of beta cell identity markers, reduced DNA damage and beta cell senescence, and increased PD-L1 within the islet microenvironment. This beta cell phenotype correlates with anti-inflammatory and exhausted immune cell states in the NOD islet. Together, these findings indicate that CR improves glucose homeostasis and remodels the islet microenvironment to promote beta cell longevity via a pro-tolerogenic immune microenvironment that reduces the risk for autoimmune diabetes.

cell biology↗

bioRxiv2025

Calorie Restriction modulates beta cell IP3R activity to regulate Ca2+ homeostasis and cell network connectivity

Calorie restriction (CR) promotes beta cell longevity by regulating cell identity, organelle and protein homeostasis, and metabolism pathways. CR beta cells have higher cAMP levels and mitochondria with an elevated potential to generate ATP. However, CR beta cells have reduced insulin secretion due to increased peripheral insulin sensitivity. How CR impacts beta cell Ca2+ homeostasis to regulate beta cell insulin release remains unknown. We investigated this question using acute pancreatic tissue slices prepared from ad-libitum (AL) or CR mice loaded with a low affinity Ca2+ indicator and recorded cytosolic Ca2+ gradients with fast confocal imaging. We exposed these slices to increasing glucose concentrations and applied our semi-automatic analysis pipeline to detect thousands of individual beta cells followed by identification of individual Ca2+ spiking events. We observed that CR beta cells have fast short-amplitude Ca2+ oscillations that correlate with largely disconnected beta cell networks across the islet. Using acetylcholine stimulation, we found that faster IP3R-driven Ca2+ oscillations linked to higher cytosolic cAMP levels protect beta cells against acute depletion of ER Ca2+ stress. Therefore, this study provides new mechanistic insight into adaptation of beta cell and of beta cell networks to CR interventions. Article highlightsO_LIBeta cells from calorie restricted (CR) mice have decreased insulin release, however the mechanisms underlying this adaptive response remain unknown. C_LIO_LICR beta cells have elevated basal cytosolic cAMP ([cAMP]cyt) compared to beta cells in control ad libitum fed (AL) mice, and they operate with faster and shorter cytosolic Ca2+ oscillations. C_LIO_LIWhile AL beta cells form interconnected activity networks, CR beta cells are largely disconnected and fire more independently of each other. C_LIO_LIIslets of CR mice can sustain prolonged activity during ER stressing conditions due to elevated IP3R activity and improved Ca2+ homeostasis. C_LI Why did we undertake this study?We have previously shown that calorie restriction (CR) promotes beta cell longevity by enhancing beta cell identity and organelle homeostasis mechanisms. This long-lived phenotype correlated with the onset of enhanced peripheral insulin sensitivity and reduced beta cell insulin release in vivo despite higher cAMP levels and increased potential for mitochondrial ATP generation. However, the mechanisms underlying the reduced cell insulin release phenotype of CR beta cells remains unknown. Therefore, we investigated the underlying Ca2+ homeostasis mechanisms regulating insulin release in AL and CR beta cells. What is the specific question(s) we wanted to answer?We were interested in determining what are the cell Ca2+ activity patterns during basal and glucose-stimulated conditions in AL and CR beta cells. In addition, we also investigated how CR beta cells respond to epinephrine inhibition and supra-stimulatory concentrations of acetylcholine (ACh), which drive acute beta cell stress by disrupting normal cAMP and ER Ca2+ signaling, respectively. Finally, we investigate whether CR beta cells formed more interconnected beta cell networks driven by changes in Ca2+ activity patterns. What did we find?We found that CR beta cells are more active with significantly higher rates of Ca2+ oscillation at basal and high glucose concentrations. In fact, CR beta cells have shorter inter-Ca2+ event intervals that are more resistant to depletion of cAMP by epinephrine application. In contrast, stimulation of IP3R activity (to force depletion of ER Ca2+ stores) by supraphysiological ACh concentrations revealed that CR beta cells were able to sustain a prolonged Ca2+ activity versus AL beta cells. Surprisingly, this enhanced beta cell activity profile reduced beta cell activity network connectivity. What are the implications of our findings?Our work demonstrates that CR beta cells have higher baseline and glucose-stimulated Ca2+ activity due to higher cAMP levels. These cells also have dominant IP3R activity that grants improved ER Ca2+ homeostasis and significantly reduces beta cell network connectivity to tone down insulin secretion. These studies provide a mechanistic understanding of how beta cells adapt to CR and to CR-associated enhanced insulin sensitivity.

cell biology↗

bioRxiv2025

Calorie restriction suppresses aging alpha cell pro-inflammatory signaling

Aging increases risk for type 2 diabetes (T2D), partly through loss of beta cell identity and function driven by metabolic stress and islet inflammation. While calorie restriction (CR) promotes beta cell longevity in young animals, its impact on cellular aging and inflammatory burden in older individuals is unclear. Using SCENIC regulon and multiomic analyses, we find that aging human alpha cells adopt a coordinated inflammatory phenotype marked by IFN-{gamma} signaling to increase major histocompatibility complex (MHC) class I presentation, and CD8+ T cell recruitment and activation towards islets. In T2D, CD8+ T cells further progress towards an effector memory state. CR reduces alpha cell MHC-I expression while subsequently suppressing CD8+ effector status and accompanied by reduced islet inflammation and immune cell infiltration in mice. Together, these findings highlight an alpha cell-immune signaling axis in aging and T2D that may underlie fibrosis and disease pathophysiology.

physiology↗

bioRxiv2025

Overnutrition directly impairs thyroid hormone biosynthesis and utilization, causing hypothyroidism, despite remarkable thyroidal adaptations

Thyroid hormones (THs: T3 and T4) are key regulators of metabolic rate and nutrient metabolism. They are controlled centrally and peripherally in a coordinated manner to elegantly match T3-mediated energy expenditure (EE) to energy availability. Hypothyroidism reduces EE and has long been blamed for obesity; however, emerging evidence suggests that, instead, obesity may drive thyroid dysfunction. Thus, we used a mouse model of diet-induced obesity to determine its direct effects on thyroid histopathology and function, deiodinase activity, and T3 action. Strikingly, overnutrition induced hypothyroidism within 3 weeks. Levels of thyroidal THs and their precursor protein thyroglobulin decreased, and ER stress was induced, indicating that thyroid function was directly impaired. We also observed pronounced histological and vascular expansion in the thyroid. Overnutrition additionally suppressed T4 activation, rendering the mice resistant to T4 and reducing EE. Our findings collectively show that overnutrition deals a double strike to TH biosynthesis and action, despite large efforts to adapt--but, fortunately, thyroid dysfunction in mice can be reversed by weight loss. In humans, BMI correlated with thyroidal vascularization, importantly demonstrating preliminary translatability. These studies lay the groundwork for novel obesity therapies that tackle hypothyroidism--which are much-needed, as no current obesity treatment works for everyone.

physiology↗

bioRxiv2025

Linking molecular pathways and islet cell dysfunction in human type 1 diabetes

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of most insulin-producing {beta}-cells, along with dysregulated glucagon secretion from pancreatic -cells. We conducted an integrated analysis that combines electrophysiological and transcriptomic profiling, along with machine learning, of islet cells from T1D donors to investigate the mechanisms underlying their dysfunction. Surviving {beta}-cells exhibit altered electrophysiological properties and transcriptomic signatures indicative of increased antigen presentation, metabolic reprogramming, and impaired protein translation. In -cells, we observed hyper-responsiveness and increased exocytosis, which are associated with upregulated immune signaling, disrupted transcription factor localization and lysosome homeostasis, as well as dysregulation of mTORC1 complex signaling. Notably, key genetic risk signals for T1D were enriched in transcripts related to -cell dysfunction, including MHC class I which were closely linked with -cell dysfunction. Our data provide novel insights into the molecular underpinnings of islet cell dysfunction in T1D, highlighting pathways that may be leveraged to preserve residual {beta}-cell function and modulate -cell activity. These findings underscore the complex interplay between immune signaling, metabolic stress, and cellular identity in shaping islet cell phenotypes in T1D. HighlightsO_LISurviving {beta}-cells in T1D show disrupted electrical function linked to metabolic reprogramming and immune stress. C_LIO_LITranscripts associated with -cell dysfunction are enriched in genetic risk alleles for T1D. C_LIO_LIUpregulated MHC class I and impaired nuclear localization of key transcription factors associate with -cell dysfunction in T1D. C_LIO_LIT1D -cells exhibit increased hyper-activity, lysosomal imbalance and impaired mTORC1 signaling, which promotes dysregulated glucagon secretion. C_LI

cell biology↗

bioRxiv2024

ER-phagy drives age-onset remodeling of endoplasmic reticulum structure-function and lifespan

The endoplasmic reticulum (ER) comprises an array of structurally distinct subdomains, each with characteristic functions. While altered ER-associated processes are linked to age-onset pathogenesis, whether shifts in ER morphology underlie these functional changes is unclear. We report that ER remodeling is a conserved feature of the aging process in models ranging from yeast to C. elegans and mammals. Focusing on C. elegans as an exemplar of metazoan aging, we find that as animals age, ER mass declines in virtually all tissues and ER morphology shifts from rough sheets to tubular ER. The accompanying large-scale shifts in proteomic composition correspond to the ER turning from protein synthesis to lipid metabolism. To drive this substantial remodeling, ER-phagy is activated early in adulthood, promoting turnover of rough ER in response to rises in luminal protein-folding burden and reduced global protein synthesis. Surprisingly, ER remodeling is a pro-active and protective response during aging, as ER-phagy impairment limits lifespan in yeast and diverse lifespan-extending paradigms promote profound remodeling of ER morphology even in young animals. Altogether our results reveal ER-phagy and ER morphological dynamics as pronounced, underappreciated mechanisms of both normal aging and enhanced longevity.

cell biology↗

bioRxiv2023

Caloric restriction promotes beta cell longevity and delays aging and senescence by enhancing cell identity and homeostasis mechanisms.

Caloric restriction (CR) extends organismal lifespan and health span by improving glucose homeostasis mechanisms. How CR affects organellar structure and function of pancreatic beta cells over the lifetime of the animal remains unknown. Here, we used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis link this transcriptional phenotype to transcription factors involved in beta cell identity (Mafa) and homeostasis (Atf6). Imaging metabolomics further demonstrates that CR beta cells are more energetically competent. In fact, high-resolution light and electron microscopy indicates that CR reduces beta cell mitophagy and increases mitochondria mass, increasing mitochondrial ATP generation. Finally, we show that long-term CR delays the onset of beta cell aging and senescence to promote longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cells during aging and diabetes.

cell biology↗

bioRxiv2021

Single-cell transcriptomics reveals a conserved metaplasia program in pancreatic injury

BACKGROUND & AIMSAcinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression. METHODSAcinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate upon injury. Transcripts of over 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared to gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining. RESULTSscRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison to KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype. CONCLUSIONSUnder conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. KrasG12D expression is sufficient to drive neoplasia from injury-induced ADM and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.

cell biology↗

bioRxiv2021

Heterogenous impairment of α-cell function in type 2 diabetes is linked to cell maturation state

In diabetes, glucagon secretion from pancreatic -cells is dysregulated. We examined -cells from human donors and mice using combined electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses -cell exocytosis by reducing P/Q-type Ca2+ channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat-feeding of mice, -cells shift towards a {beta}-cell-like electrophysiologic profile in concert with an up-regulation of the {beta}-cell Na+ channel isoform Scn9a and indications of impaired -cell identity. In human -cells we identify links between cell membrane properties and cell surface signalling receptors, mitochondrial respiratory complex assembly, and cell maturation. Cell type classification using machine learning of electrophysiology data demonstrates a heterogenous loss of electrophysiologic identity in -cells from donors with T2D. Indeed, a sub-set of -cells with impaired exocytosis is defined by an enrichment in progenitor markers suggesting important links between -cell maturation state and dysfunction in T2D. Key findingsO_LI-cell exocytosis is suppressed by glucose-dependent inhibition of P/Q-type Ca2+ currents C_LIO_LIDysfunction of -cells in type 2 diabetes is associated with a {beta}-cell-like electrophysiologic signature C_LIO_LIPatch-seq links maturation state, the mitochondrial respiratory chain, and cell surface receptor expression to -cell function C_LIO_LI-cell dysfunction occurs preferentially in cells enriched in endocrine lineage markers C_LI

cell biology↗

bioRxiv2019

Aging of human endocrine pancreatic cell types is heterogeneous and sex-specific

The human endocrine pancreas must regulate glucose homeostasis throughout the human lifespan, which is generally decades. We performed meta-analysis of single-cell, RNA-sequencing datasets derived from 36 individuals, as well as functional analyses, to characterize age-associated changes to the major endocrine pancreatic cell types. Increasing age was associated with shifts in pancreatic alpha and beta cell identity and loss of nuclear integrity in non-diabetic humans. In non-diabetic individuals [≥] 50 years old, 80% of their beta cells exhibited a transcriptional signature similar to cells from type-2 diabetic (T2D) donors. Surprisingly, [~]5% of beta cells from T2D donors retained a youthful, N.D. transcriptional profile. Furthermore, beta cell function was reduced by 50% during aging in men but not women, which may explain sex-associated differences in diabetes etiology. These analyses reveal that aging of the human endocrine pancreas is sex- and cell-type specific.

physiology↗