The age-dependent regulation of pancreatic islet landscape is fueled by a HNF1a-immune signaling loop.

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.

Molecular profiling of NOD mouse islets reveals a novel regulator of insulitis onset.

Non-obese diabetes (NOD) mice are an established, spontaneous model of type 1 diabetes in which diabetes develops through insulitis. Using next-generation sequencing, coupled with pathway analysis, the molecular fingerprint of early insulitis was mapped in a cohort of mice ranging from 4 to 12 weeks of age. The resulting dynamic timeline revealed an initial decrease in proliferative capacity followed by the emergence of an inflammatory signature between 6 and 8 weeks that increased to a regulatory plateau between 10 and 12 weeks. The inflammatory signature is identified by the activation of central immunogenic factors such as Infg, Il1b, and Tnfa, and activation of canonical inflammatory signaling. Analysis of the regulatory landscape revealed the transcription factor Atf3 as a potential novel modulator of inflammatory signaling in the NOD islets. Furthermore, the Hedgehog signaling pathway correlated with Atf3 regulation, suggesting that the two play a role in regulating islet inflammation; however, further studies are needed to establish the nature of this connection.

Cell identity dynamics and insight into insulin secretagogues when employing stem cell-derived islets for disease modeling.

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.

Glucose Concentration in Regulating Induced Pluripotent Stem Cells Differentiation Toward Insulin-Producing Cells.

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.

The GLI code controls HNF1A levels during foregut differentiation.

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.

Circadian organization of lipid landscape is perturbed in type 2 diabetic patients.

Lipid homeostasis in humans follows a diurnal pattern in muscle and pancreatic islets, altered upon metabolic dysregulation. We employ tandem and liquid-chromatography mass spectrometry to investigate daily regulation of lipid metabolism in subcutaneous white adipose tissue (SAT) and serum of type 2 diabetic (T2D) and non-diabetic (ND) human volunteers (n = 12). Around 8% of ≈440 lipid metabolites exhibit diurnal rhythmicity in serum and SAT from ND and T2D subjects. The spectrum of rhythmic lipids differs between ND and T2D individuals, with the most substantial changes observed early morning, as confirmed by lipidomics in an independent cohort of ND and T2D subjects (n = 32) conducted at a single morning time point. Strikingly, metabolites identified as daily rhythmic in both serum and SAT from T2D subjects exhibit phase differences. Our study reveals massive temporal and tissue-specific alterations of human lipid homeostasis in T2D, providing essential clues for the development of lipid biomarkers in a temporal manner.

Global proteomics reveals insulin abundance as a marker of human islet homeostasis alterations.

AIM: The variation in quality between the human islet samples represents a major problem for research, especially when used as control material. The assays assessing the quality of human islets used in research are non-standardized and limited, with many important parameters not being consistently assessed. High-throughput studies aimed at characterizing the diversity and segregation markers among apparently functionally healthy islet preps are thus a requirement. Here, we designed a pilot study to comprehensively identify the diversity of global proteome signatures and the deviation from normal homeostasis in randomly selected human-isolated islet samples. METHODS: By using Tandem Mass Tag 16-plex proteomics, we focused on the recurrently observed disparity in the detected insulin abundance between the samples, used it as a segregating parameter, and analyzed the correlated changes in the proteome signature and homeostasis by pathway analysis. RESULTS: In this pilot study, we showed that insulin protein abundance is a predictor of human islet homeostasis and quality. This parameter is independent of other quality predictors within their acceptable range, thus being able to further stratify islets samples of apparent good quality. Human islets with low amounts of insulin displayed changes in their metabolic and signaling profile, especially in regard to energy homeostasis and cell identity maintenance. We further showed that xenotransplantation into diabetic hosts is not expected to improve the pre-transplantation signature, as it has a negative effect on energy balance, antioxidant activity, and islet cell identity. CONCLUSIONS: Insulin protein abundance predicts significant changes in human islet homeostasis among random samples of apparently good quality.

Stem cells: The cell that does it all.

How do animals replace all their worn-out cells to maintain their tissues? A new study shows that, in the cnidarian Hydractinia symbiolongicarpus, a single adult stem cell is sufficient to generate the entire repertoire of somatic and germ line cells.

Modulation of Unfolded Protein Response Restores Survival and Function of ß-Cells Exposed to the Endocrine Disruptor Bisphenol A.

Diabetes is a metabolic disease that currently affects nearly half a billion people worldwide. ß-cells dysfunction is one of the main causes of diabetes. Exposure to endocrine-disrupting chemicals is correlated with increased diabetes incidence. We hypothesized that treatment with bisphenol A (BPA) induces endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR), leading to impaired function of the ß-cells, which over time, can cause diabetes. In this study, we aimed to evaluate UPR pathways activation under BPA treatment in ß-cells and possible recovery of ER homeostasis. MIN6 cells (mouse insulinoma cell line) and isolated pancreatic islets from NOR (non-obese diabetes resistant) mice were treated with BPA. We analyzed the impact of BPA on ß-cell viability, the architecture of the early secretory pathway, the synthesis and processing of insulin and the activation of UPR sensors and effectors. We found that the addition of the chemical chaperone TUDCA rescues the deleterious effects of BPA, resulting in improved viability, morphology and function of the ß-cells. In conclusion, we propose that modulators of UPR can be used as therapeutic interventions targeted towards regaining ß-cells homeostasis.

Type 2 diabetes disrupts circadian orchestration of lipid metabolism and membrane fluidity in human pancreatic islets.

Recent evidence suggests that circadian clocks ensure temporal orchestration of lipid homeostasis and play a role in pathophysiology of metabolic diseases in humans, including type 2 diabetes (T2D). Nevertheless, circadian regulation of lipid metabolism in human pancreatic islets has not been explored. Employing lipidomic analyses, we conducted temporal profiling in human pancreatic islets derived from 10 nondiabetic (ND) and 6 T2D donors. Among 329 detected lipid species across 8 major lipid classes, 5% exhibited circadian rhythmicity in ND human islets synchronized in vitro. Two-time point-based lipidomic analyses in T2D human islets revealed global and temporal alterations in phospho- and sphingolipids. Key enzymes regulating turnover of sphingolipids were rhythmically expressed in ND islets and exhibited altered levels in ND islets bearing disrupted clocks and in T2D islets. Strikingly, cellular membrane fluidity, measured by a Nile Red derivative NR12S, was reduced in plasma membrane of T2D diabetic human islets, in ND donors' islets with disrupted circadian clockwork, or treated with sphingolipid pathway modulators. Moreover, inhibiting the glycosphingolipid biosynthesis led to strong reduction of insulin secretion triggered by glucose or KCl, whereas inhibiting earlier steps of de novo ceramide synthesis resulted in milder inhibitory effect on insulin secretion by ND islets. Our data suggest that circadian clocks operative in human pancreatic islets are required for temporal orchestration of lipid homeostasis, and that perturbation of temporal regulation of the islet lipid metabolism upon T2D leads to altered insulin secretion and membrane fluidity. These phenotypes were recapitulated in ND islets bearing disrupted clocks.

Islet cell replacement and transplantation immunology in a mouse strain with inducible diabetes.

Improved models of experimental diabetes are needed to develop cell therapies for diabetes. Here, we introduce the B6 RIP-DTR mouse, a model of experimental diabetes in fully immunocompetent animals. These inbred mice harbor the H2b major histocompatibility complex (MHC), selectively express high affinity human diphtheria toxin receptor (DTR) in islet ß-cells, and are homozygous for the Ptprca (CD45.1) allele rather than wild-type Ptprcb (CD45.2). 100% of B6 RIP-DTR mice rapidly became diabetic after a single dose of diphtheria toxin, and this was reversed indefinitely after transplantation with islets from congenic C57BL/6 mice. By contrast, MHC-mismatched islets were rapidly rejected, and this allotransplant response was readily monitored via blood glucose and graft histology. In peripheral blood of B6 RIP-DTR with mixed hematopoietic chimerism, CD45.2 BALB/c donor blood immune cells were readily distinguished from host CD45.1 cells by flow cytometry. Reliable diabetes induction and other properties in B6 RIP-DTR mice provide an important new tool to advance transplant-based studies of islet replacement and immunomodulation to treat diabetes.

Mapping Proteome Changes in Microsatellite Stable, Recurrent Colon Cancer Reveals a Significant Immune System Signature.

BACKGROUND/AIM: Better stratification of the risk of relapse will help select the right patients for adjuvant treatment and improve targeted therapies for patients with colon cancer. MATERIALS AND METHODS: To understand why a subset of tumors relapse, we compared the proteome of two groups of patients with colon cancer with similar stage, stratified based on the presence or absence of recurrence. RESULTS: Using tumor biopsies from the primary operation, we identified dissimilarity between recurrent and nonrecurrent mismatch satellite stable colon cancer and found that signaling related to immune activation and inflammation was associated with relapse. CONCLUSION: Immune modulation may have an effect on mismatch satellite stable colon cancer. At present, immune therapy is offered primarily to microsatellite instable colon cancer. Hopefully, immune therapy in mismatch satellite stable colon cancer beyond PD-1 and PD-L1 inhibitors can be implemented.

Spatial Environment Affects HNF4A Mutation-Specific Proteome Signatures and Cellular Morphology in hiPSC-Derived ß-Like Cells.

Studies of monogenic diabetes are particularly useful because we can gain insight into the molecular events of pancreatic ß-cell failure. Maturity-onset diabetes of the young 1 (MODY1) is a form of monogenic diabetes caused by a mutation in the HNF4A gene. Human-induced pluripotent stem cells (hiPSCs) provide an excellent tool for disease modeling by subsequently directing differentiation toward desired pancreatic islet cells, but cellular phenotypes in terminally differentiated cells are notoriously difficult to detect. Re-creating a spatial (three-dimensional [3D]) environment may facilitate phenotype detection. We studied MODY1 by using hiPSC-derived pancreatic ß-like patient and isogenic control cell lines in two different 3D contexts. Using size-adjusted cell aggregates and alginate capsules, we show that the 3D context is critical to facilitating the detection of mutation-specific phenotypes. In 3D cell aggregates, we identified irregular cell clusters and lower levels of structural proteins by proteome analysis, whereas in 3D alginate capsules, we identified altered levels of glycolytic proteins in the glucose sensing apparatus by proteome analysis. Our study provides novel knowledge on normal and abnormal function of HNF4A, paving the way for translational studies of new drug targets that can be used in precision diabetes medicine in MODY.

A Method for Encapsulation and Transplantation into Diabetic Mice of Human Induced Pluripotent Stem Cells (hiPSC)-Derived Pancreatic Progenitors.

Pancreatic islet endocrine cells generated from patient-derived induced pluripotent stem cells represent a great strategy for both disease modeling and regenerative medicine. Nevertheless, these cells inherently miss the effects of the intricate network of systemic signals characterizing the living organisms. Xenotransplantation of in vitro differentiating cells into murine hosts substantially compensates for this drawback.Here we describe our transplantation strategy of encapsulated differentiating pancreatic progenitors into diabetic immunosuppressed (NSG) overtly diabetic mice generated by the total ablation of insulin-producing cells following diphtheria toxin administration. We will detail the differentiation protocol employed, the alginate encapsulation procedure, and the xenotransplantation steps required for a successful and reproducible experiment.

Stage-specific transcriptomic changes in pancreatic α-cells after massive ß-cell loss.

BACKGROUND: Loss of pancreatic insulin-secreting ß-cells due to metabolic or autoimmune damage leads to the development of diabetes. The discovery that α-cells can be efficiently reprogrammed into insulin-secreting cells in mice and humans has opened promising avenues for innovative diabetes therapies. ß-cell loss triggers spontaneous reprogramming of only 1-2% of α-cells, limiting the extent of regeneration. Most α-cells are refractory to conversion and their global transcriptomic response to severe ß-cell loss as well as the mechanisms opposing their reprogramming into insulin producers are largely unknown. Here, we performed RNA-seq on FAC-sorted α-cells to characterize their global transcriptional responses at different time points after massive ß-cell ablation. RESULTS: Our results show that α-cells undergo stage-specific transcriptional changes 5- and 15-days post-diphtheria toxin (DT)-mediated ß-cell ablation. At 5 days, α-cells transiently upregulate various genes associated with interferon signaling and proliferation, including Interferon Induced Protein with Tetratricopeptide Repeats 3 (Ifit3). Subsequently, at 15 days post ß-cell ablation, α-cells undergo a transient downregulation of genes from several pathways including Insulin receptor, mTOR and MET signaling. CONCLUSIONS: The results presented here pinpoint novel markers discriminating α-cells at different stages after acute ß-cell loss, and highlight additional signaling pathways that are modulated in α-cells in this context.

Chronically Elevated Exogenous Glucose Elicits Antipodal Effects on the Proteome Signature of Differentiating Human iPSC-Derived Pancreatic Progenitors.

The past decade revealed that cell identity changes, such as dedifferentiation or transdifferentiation, accompany the insulin-producing ß-cell decay in most diabetes conditions. Mapping and controlling the mechanisms governing these processes is, thus, extremely valuable for managing the disease progression. Extracellular glucose is known to influence cell identity by impacting the redox balance. Here, we use global proteomics and pathway analysis to map the response of differentiating human pancreatic progenitors to chronically increased in vitro glucose levels. We show that exogenous high glucose levels impact different protein subsets in a concentration-dependent manner. In contrast, regardless of concentration, glucose elicits an antipodal effect on the proteome landscape, inducing both beneficial and detrimental changes in regard to achieving the desired islet cell fingerprint. Furthermore, we identified that only a subgroup of these effects and pathways are regulated by changes in redox balance. Our study highlights a complex effect of exogenous glucose on differentiating pancreas progenitors characterized by a distinct proteome signature.

The core clock transcription factor BMAL1 drives circadian ß-cell proliferation during compensatory regeneration of the endocrine pancreas.

Circadian clocks in pancreatic islets participate in the regulation of glucose homeostasis. Here we examined the role of these timekeepers in ß-cell regeneration after the massive ablation of ß cells by doxycycline-induced expression of diphtheria toxin A (DTA) in Insulin-rtTA/TET-DTA mice. Since we crossed reporter genes expressing α- and ß-cell-specific fluorescent proteins into these mice, we could follow the fate of α- and ß cells separately. As expected, DTA induction resulted in an acute hyperglycemia, which was accompanied by dramatic changes in gene expression in residual ß cells. In contrast, only temporal alterations of gene expression were observed in α cells. Interestingly, ß cells entered S phase preferentially during the nocturnal activity phase, indicating that the diurnal rhythm also plays a role in the orchestration of ß-cell regeneration. Indeed, in arrhythmic Bmal1-deficient mice, which lack circadian clocks, no compensatory ß-cell proliferation was observed, and the ß-cell ablation led to aggravated hyperglycemia, hyperglucagonemia, and fatal diabetes.

Bioinformatic Analyses of miRNA-mRNA Signature during hiPSC Differentiation towards Insulin-Producing Cells upon HNF4α Mutation.

Mutations in the hepatocyte nuclear factor 4α (HNF4α) gene affect prenatal and postnatal pancreas development, being characterized by insulin-producing ß-cell dysfunction. Little is known about the cellular and molecular mechanisms leading to ß-cell failure as result of HNF4α mutation. In this study, we compared the miRNA profile of differentiating human induced pluripotent stem cells (hiPSC) derived from HNF4α+/Δ mutation carriers and their family control along the differentiation timeline. Moreover, we associated this regulation with the corresponding transcriptome profile to isolate transcript-miRNA partners deregulated in the mutated cells. This study uncovered a steep difference in the miRNA regulation pattern occurring during the posterior foregut to pancreatic endoderm transition, defining early and late differentiation regulatory windows. The pathway analysis of the miRNAome-transcriptome interactions revealed a likely gradual involvement of HNF4α+/Δ mutation in p53-mediated cell cycle arrest, with consequences for the proliferation potential, survival and cell fate acquisition of the differentiating cells. The present study is based on bioinformatics approaches and we expect that, pending further experimental validation, certain miRNAs deregulated in the HNF4α+/Δ cells would prove useful for therapy.

In vivo Environment Swiftly Restricts Human Pancreatic Progenitors Toward Mono-Hormonal Identity via a HNF1A/HNF4A Mechanism.

Generating insulin-producing ß-cells from human induced pluripotent stem cells is a promising cell replacement therapy for improving or curing insulin-dependent diabetes. The transplantation of end-stages differentiating cells into living hosts was demonstrated to improve ß-cell maturation. Nevertheless, the cellular and molecular mechanisms outlining the transplanted cells' response to the in vivo environment are still to be properly characterized. Here we use global proteomics and large-scale imaging techniques to demultiplex and filter the cellular processes and molecular signatures modulated by the immediate in vivo effect. We show that in vivo exposure swiftly confines in vitro generated human pancreatic progenitors to single hormone expression. The global proteome landscape of the transplanted cells was closer to native human islets, especially in regard to energy metabolism and redox balance. Moreover, our study indicates a possible link between these processes and certain epigenetic regulators involved in cell identity. Pathway analysis predicted HNF1A and HNF4A as key regulators controlling the in vivo islet-promoting response, with experimental evidence suggesting their involvement in confining islet cell fate following xeno-transplantation.