Loss Of Β-cell Identity Research Articles

Loss of β-cell identity and dedifferentiation, not an irreversible process?

Type 2 diabetes (T2D) is a polygenic metabolic disorder characterized by insulin resistance in peripheral tissues and impaired insulin secretion by the pancreas. While the decline in insulin production and secretion was previously attributed to apoptosis of insulin-producing β-cells, recent studies indicate that β-cell apoptosis rates are relatively low in diabetes. Instead, β-cells primarily undergo dedifferentiation, a process where they lose their specialized identity and transition into non-functional endocrine progenitor-like cells, ultimately leading to β-cell failure. The underlying mechanisms driving β-cell dedifferentiation remain elusive due to the intricate interplay of genetic factors and cellular stress. Understanding these mechanisms holds the potential to inform innovative therapeutic approaches aimed at reversing β-cell dedifferentiation in T2D. This review explores the proposed drivers of β-cell dedifferentiation leading to β-cell failure, and discusses current interventions capable of reversing this process, thus restoring β-cell identity and function.

Pancreatic β-Cell Identity Change through the Lens of Single-Cell Omics Research.

The main hallmark in the development of both type 1 and type 2 diabetes is a decline in functional β-cell mass. This decline is predominantly attributed to β-cell death, although recent findings suggest that the loss of β-cell identity may also contribute to β-cell dysfunction. This phenomenon is characterized by a reduced expression of key markers associated with β-cell identity. This review delves into the insights gained from single-cell omics research specifically focused on β-cell identity. It highlights how single-cell omics based studies have uncovered an unexpected level of heterogeneity among β-cells and have facilitated the identification of distinct β-cell subpopulations through the discovery of cell surface markers, transcriptional regulators, the upregulation of stress-related genes, and alterations in chromatin activity. Furthermore, specific subsets of β-cells have been identified in diabetes, such as displaying an immature, dedifferentiated gene signature, expressing significantly lower insulin mRNA levels, and expressing increased β-cell precursor markers. Additionally, single-cell omics has increased insight into the detrimental effects of diabetes-associated conditions, including endoplasmic reticulum stress, oxidative stress, and inflammation, on β-cell identity. Lastly, this review outlines the factors that may influence the identification of β-cell subpopulations when designing and performing a single-cell omics experiment.

Pancreatic islet cell plasticity: Pathogenic or therapeutically exploitable?

The development of pancreatic islet endocrine cells is a tightly regulated process leading to the generation of distinct cell types harbouring different hormones in response to small changes in environmental stimuli. Cell differentiation is driven by transcription factors that are also critical for the maintenance of the mature islet cell phenotype. Alteration of the insulin-secreting β-cell transcription factor set by prolonged metabolic stress, associated with the pathogenesis of diabetes, obesity or pregnancy, results in the loss of β-cell identity through de- or transdifferentiation. Importantly, the glucose-lowering effects of approved and experimental antidiabetic agents, including glucagon-like peptide-1 mimetics, novel peptides and small molecules, have been associated with preventing or reversing β-cell dedifferentiation or promoting the transdifferentiation of non-β-cells towards an insulin-positive β-cell-like phenotype. Therefore, we review the manifestations of islet cell plasticity in various experimental settings and discuss the physiological and therapeutic sides of this phenomenon, focusing on strategies for preventing β-cell loss or generating new β-cells in diabetes. A better understanding of the molecular mechanisms underpinning islet cell plasticity is a prerequisite for more targeted therapies to help prevent β-cell decline in diabetes.

M6 A mRNA methylation: Biological features, mechanisms, and therapeutic potentials in type 2 diabetes mellitus.

As the most common internal post-transcriptional RNA modification in eukaryotic cells, N6-methyladenosine (m6 A) performs a dynamic and reversible role in a variety of biological processes mediated by methyltransferases (writers), demethylases (erasers), and m6 A binding proteins (readers). M6 A methylation enables transcriptome conversion in different signals that regulate various physiological activities and organ development. Over the past few years, emerging studies have identified that mRNA m6 A regulators defect in β-cell leads to abnormal regulation of the target mRNAs, thereby resulting in β-cell dysfunction and loss of β-cell identity and mass, which are strongly associated with type 2 diabetes mellitus (T2DM) pathogenesis. Also, mRNA m6 A modification has been implicated with insulin resistance in muscles, fat, and liver cells/tissues. In this review, we elaborate on the biological features of m6 A methylation; provide a comprehensive overview of the underlying mechanisms that how it controls β-cell function, identity, and mass as well as insulin resistance; highlight its connections to glucose metabolism and lipid metabolism linking to T2DM; and further discuss its role in diabetes complications and its therapeutic potentials for T2DM diagnosis and treatment.

Pharmacological HDAC inhibition impairs pancreatic β-cell function through an epigenome-wide reprogramming

Histone deacetylases enzymes (HDACs) are chromatin modifiers that regulate gene expression through deacetylation of lysine residues within specific histone and non-histone proteins. A cell-specific gene expression pattern defines the identity of insulin-producing pancreatic β cells, yet molecular networks driving this transcriptional specificity are not fully understood. Here, we investigated the HDAC-dependent molecular mechanisms controlling pancreatic β-cell identity and function using the pan-HDAC inhibitor trichostatin A through chromatin immunoprecipitation assays and RNA sequencing experiments. We observed that TSA alters insulin secretion associated with β-cell specific transcriptome programming in both mouse and human β-cell lines, as well as on human pancreatic islets. We also demonstrated that this alternative β-cell transcriptional program in response to HDAC inhibition is related to an epigenome-wide remodeling at both promoters and enhancers. Our data indicate that HDAC activity could be required to protect against loss of β-cell identity with unsuitable expression of genes associated with alternative cell fates.

Deficiency of the metabolic enzyme SCHAD in pancreatic β-cells promotes amino acid–sensitive hypoglycemia

Congenital hyperinsulinism of infancy (CHI) can be caused by a deficiency of the ubiquitously expressed enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD). To test the hypothesis that SCHAD-CHI arises from a specific defect in pancreatic β-cells, we created genetically engineered β-cell-specific (β-SKO) or hepatocyte-specific (L-SKO) SCHAD knockout mice. While L-SKO mice were normoglycemic, plasma glucose in β-SKO animals was significantly reduced in the random-fed state, after overnight fasting, and following refeeding. The hypoglycemic phenotype was exacerbated when the mice were fed a diet enriched in leucine, glutamine, and alanine. Intraperitoneal injection of these three amino acids led to a rapid elevation in insulin levels in β-SKO mice compared to controls. Consistently, treating isolated β-SKO islets with the amino acid mixture potently enhanced insulin secretion compared to controls in a low-glucose environment. RNA sequencing of β-SKO islets revealed reduced transcription of β-cell identity genes and upregulation of genes involved in oxidative phosphorylation, protein metabolism, and Ca2+ handling. The β-SKO mouse offers a useful model to interrogate the intra-islet heterogeneity of amino acid sensing given the very variable expression levels of SCHAD within different hormonal cells, with high levels in β- and δ-cells and virtually absent α-cell expression. We conclude that the lack of SCHAD protein in β-cells results in a hypoglycemic phenotype characterized by increased sensitivity to amino acid-stimulated insulin secretion and loss of β-cell identity.

Maladaptive positive feedback production of ChREBPβ underlies glucotoxic β-cell failure

Preservation and expansion of β-cell mass is a therapeutic goal for diabetes. Here we show that the hyperactive isoform of carbohydrate response-element binding protein (ChREBPβ) is a nuclear effector of hyperglycemic stress occurring in β-cells in response to prolonged glucose exposure, high-fat diet, and diabetes. We show that transient positive feedback induction of ChREBPβ is necessary for adaptive β-cell expansion in response to metabolic challenges. Conversely, chronic excessive β-cell-specific overexpression of ChREBPβ results in loss of β-cell identity, apoptosis, loss of β-cell mass, and diabetes. Furthermore, β-cell “glucolipotoxicity” can be prevented by deletion of ChREBPβ. Moreover, ChREBPβ-mediated cell death is mitigated by overexpression of the alternate CHREBP gene product, ChREBPα, or by activation of the antioxidant Nrf2 pathway in rodent and human β-cells. We conclude that ChREBPβ, whether adaptive or maladaptive, is an important determinant of β-cell fate and a potential target for the preservation of β-cell mass in diabetes.

Oxidative Stress Leads to β-Cell Dysfunction Through Loss of β-Cell Identity.

Pancreatic β-cell failure is a critical event in the onset of both main types of diabetes mellitus but underlying mechanisms are not fully understood. β-cells have low anti-oxidant capacity, making them more susceptible to oxidative stress. In type 1 diabetes (T1D), reactive oxygen species (ROS) are associated with pro-inflammatory conditions at the onset of the disease. Here, we investigated the effects of hydrogen peroxide-induced oxidative stress on human β-cells. We show that primary human β-cell function is decreased. This reduced function is associated with an ER stress response and the shuttling of FOXO1 to the nucleus. Furthermore, oxidative stress leads to loss of β-cell maturity genes MAFA and PDX1, and to a concomitant increase in progenitor marker expression of SOX9 and HES1. Overall, we propose that oxidative stress-induced β-cell failure may result from partial dedifferentiation. Targeting antioxidant mechanisms may preserve functional β-cell mass in early stages of development of T1D.

175-OR: The Mitochondrial Life Cycle Determines ß-Cell Maturity through Activation of the Integrated Stress Response

Mitochondrial function is pivotal to β-cell competence. The mitochondrial life cycle balances mitochondrial biogenesis and turnover (mitophagy) to ensure optimal metabolic function. Human type 2 diabetic (T2D) β-cells are known to develop mitochondrial structural/functional defects, suggestive of a defective mitochondrial life cycle. However, it is unclear if these defects are a cause or consequence of T2D. Here, we observed that human T2D β-cells had reduced mitophagic flux, mtDNA content, and expression of mitochondrially encoded genes. To test the importance of the mitochondrial life cycle to drive β-cell failure in T2D, we developed 2 distinct β-cell specific mouse models: βTfamKO (to deplete mtDNA) and βClec16aΚΟ (to impair mitophagy). We observed age dependent loss of glucose tolerance, glucose stimulated insulin secretion and β-cell mass in both models, which was exacerbated by obesity in βClec16aΚΟ mice. Loss of β-cell mass was largely independent of changes in proliferation or apoptosis but rather, due to an induction of β-cell immaturity. Using lineage tracing approaches, we confirmed that βClec16aKO and βTfamKO mice induce formation of both insulin-negative immature β-cells and β-to-α cell transdifferentiation. Further, high-throughput gene expression profiling, biochemical, and metabolic assays highlighted that either Clec16a or Tfam deficiency induces an aberrant retrograde signaling program, manifested by reductions in cellular ATP and activation of the integrated stress response (ISR). Importantly, inhibition of the ISR in vivo ameliorated glucose intolerance, defective insulin secretion, β-cell immaturity, and loss of β-cell mass, suggesting that aberrant retrograde signaling may directly lead to loss of β-cell identity. Taken together, our studies illustrate that a unified mitochondrial lifecycle is necessary to maintain β-cell mass and identity and may be targeted to prevent β-cell failure in T2D. Disclosure G. Pearson: None. N. Lawlor: None. J. Zhu: None. E. M. Walker: None. E. C. Reck: None. M. L. Stitzel: None. S. Soleimanpour: None. Funding American Diabetes Association (1-19-PDF-063 to G.P.)

2059-P: Gene Repression during Maturation of Pancreatic ß-Cells Mirrors Those Upregulated in Islets of Diabetic db/db Mice

Pancreatic β-cells are specialized to properly regulate glucose metabolism. Loss of individual transcription factors that are critical for endocrine differentiation embryonically causes a deep loss of cell identity and upregulation of genes normally repressed in mature β-cells. We hypothesized that the factors important for maintenance of mature β-cell homeostasis are those that are lost in the identity loss or dedifferentiation. To identify genes critical for acquisition and loss of β-cell identity, we examined islets and β-cells by comprehensive gene expression analyses. Islets isolated from 8 wk (mature) and 4 wk (immature) old C57BL/6J mice were analyzed by microarray, and FACS-sorted mature β-cells (Mafa+7AAD-) and other islet cells including Mafa negative β-cells (Mafa-7AAD-) purified from Mafa reporter mice by RNAseq. We found that the maturation process involved genome-wide downregulated gene expression. Mature islets and Mafa+ β-cells had suppression of many genes including “β-cell disallowed genes” such as Igfbp4 and Nfib, but little to noupregulation of genes critical for β-cell function such as Ins1, Slc2a2, Ucn3 and Sytl4 from 4 wk to 8 wk. In contrast, most of the suppressed genes were significantly upregulated (by RNAseq) in islets from 21 wk old diabetic BKS-db/db mice compared with BKS-db/+ (misty). Comprehensive methylation screening (MeDIP-seq) of DNA from islets of 4 and 8 wk old C57BL/6J mice specifically identified highly methylated regulatory regions in some “repressed” genes in 8 wk old islets. In conclusion, the gain of identity during maturation phase of β-cells mirrors that of the dedifferentiation during diabetes. Disclosure W. Nishimura: None. M. Tumurkhuu: None. Y. Noda: None. K. Yasuda: None. Funding Japan Society for the Promotion of Science (JP16H01647, JP26461351, JP17K09843); International University of Health and Welfare; Japan Diabetes Foundation; Novartis Pharmaceuticals

Raptor determines \u03b2-cell identity and plasticity independent of hyperglycemia in mice

Compromised β-cell identity is emerging as an important contributor to β-cell failure in diabetes; however, the precise mechanism independent of hyperglycemia is under investigation. We have previously reported that mTORC1/Raptor regulates functional maturation in β-cells. In the present study, we find that diabetic β-cell specific Raptor-deficient mice (βRapKOGFP) show reduced β-cell mass, loss of β-cell identity and acquisition of α-cell features; which are not reversible upon glucose normalization. Deletion of Raptor directly impairs β-cell identity, mitochondrial metabolic coupling and protein synthetic activity, leading to β-cell failure. Moreover, loss of Raptor activates α-cell transcription factor MafB (via modulating C/EBPβ isoform ratio) and several α-cell enriched genes i.e. Etv1 and Tspan12, thus initiates β- to α-cell reprograming. The present findings highlight mTORC1 as a metabolic rheostat for stabilizing β-cell identity and repressing α-cell program at normoglycemic level, which might present therapeutic opportunities for treatment of diabetes.

334-LB: Dapagliflozin, a SGLT2 Inhibitor, Therapy Improves ß-Cell Function in Neonatal Diabetes by Reducing Oxidative and ER Stress, and Loss of ß-Cell Identity

Loss of pancreatic β-cell identity, rather than β-cell death, has been recently demonstrated in both neonatal and type-2 diabetes. However, the underlying mechanisms and temporal progression remains elusive. By using an insulin secretory-deficient KATP-GOF mouse model of human neonatal diabetes, we demonstrate normal insulin content at day 7 despite severe diabetes, but a dramatic decrease (55%) in content at day 15 of diabetes. Loss of content was accompanied by a marked increase in proinsulin protein, a striking increase in oxidative- and ER-stress markers such as ROS, TXNIP, sXBP1 and ATF6, distended ER and decreased number of insulin granules. These changes lead to later loss of β-cell identity and increased α-cell identity (ARX increased 50%), glucagon content and secretion. We hypothesized that lowering blood glucose alone will be sufficient to reverse this process and improve β-cell function. Therefore, diabetic KATP-GOF mice were treated with dapagliflozin (DAPA), a sodium-glucose transporter-2 (SGLT2) inhibitor clinically used to reduce blood glucose by preventing renal glucose reabsorption. As expected, DAPA therapy significantly reduced blood glucose without changing circulating insulin levels. Ten days DAPA therapy markedly improved insulin content, reduced proinsulin/insulin ratio and enhanced β-cell function by reducing oxidative and ER stress and preventing loss of β-cell identity. Together, we clearly demonstrate that loss of insulin content, increased oxidative and ER stress, and changes in calcium dynamics underlie later loss of β-cell identity in diabetes, and that these features can be prevented/reversed by normalization of blood glucose alone with SGLT2 inhibitors. From the therapeutic perspective, these results have important implications indicating that early alleviation of β-cell stress may prevent loss of β-cell mass and preserve islet-cell identity, architecture and function. Disclosure Z. Yan: None. Z.A. Shyr: None. M.S. Remedi: None. Funding National Institutes of Health (DK098584 to M.S.R.), (T32DK108742 to Z.A.S.)

The CCR4-NOT Complex Maintains ß-Cell Identity through the Repression of ß-Cell Disallowed Genes

Loss of β-cell identity, dedifferentiation and reprogramming are recognized as mechanisms of β-cell dysfunction in diabetes. Molecular β-cell identity is not only defined by the expression of signature functional β-cell specific genes but also the repression of several genes recently identified in β-cell research as β-cell disallowed genes. The molecular mechanisms involved in the repression of β-cell disallowed genes are largely unknown. Here, we show that the CCR4-NOT complex, a major deadenylase conserved in eukaryotes, is involved in post-transcriptional regulation of β-cell disallowed genes. β-cell specific depletion of CNOT3 (CNOT3βKO), a CCR4-NOT complex subunit, in mice promotes deceased insulin secretion, early impaired glucose tolerance and the development of overt diabetes at 12 weeks of age. CNOT3βKO islets display decreased insulin content and decreased glucose responsiveness. Furthermore, CNOT3 depleted ß cells exhibit ultrastructural abnormalities including: degranulated β cells, increased immature insulin granules and abnormal mitochondria. RNAseq and qRT-PCR analyses revealed that CNOT3βKO islets have decreased expression of β-cell functional genes: (Ins1, Ins2, MafA, Pcsk2 and Cpe) and increased expression of 9 of the 11 core β-cell disallowed genes: (Slc16A1, Ldha, Pdgfra, Cxcl12, Igfbp4, Oat, Maf, Smad3 and Cd302) and progenitor-like cells genes: (Nanog and Ngn3) supporting the loss of β-cell identity. Using the total RNAseq data, we assessed mRNA stability by calculating the relative amounts of introns and exons for a given mRNA. RNA immunoprecipitation revealed that the CCR4-NOT complex directly binds several upregulated stabilized β-cell disallowed genes: Slc16A1, Tgm2, Cat and Parp3 genes. The present data highlight deadenylation as a novel molecular mechanism regulating β-cell disallowed genes. Thus, we further propose that the CCR4-NOT complex is essential for maintaining β-cell identity by repressing β-cell disallowed genes. Disclosure D. Mostafa: None. A. Takahashi: None. A. Yanagiya: None. T. Yamamoto: None.

Increased vimentin in human α- and β-cells in type 2 diabetes.

Type 2 diabetes (T2DM) is associated with pancreatic islet dysfunction. Loss of β-cell identity has been implicated via dedifferentiation or conversion to other pancreatic endocrine cell types. How these transitions contribute to the onset and progression of T2DM in vivo is unknown. The aims of this study were to determine the degree of epithelial-to-mesenchymal transition occurring in α and β cells in vivo and to relate this to diabetes-associated (patho)physiological conditions. The proportion of islet cells expressing the mesenchymal marker vimentin was determined by immunohistochemistry and quantitative morphometry in specimens of pancreas from human donors with T2DM (n = 28) and without diabetes (ND, n = 38) and in non-human primates at different stages of the diabetic syndrome: normoglycaemic (ND, n = 4), obese, hyperinsulinaemic (HI, n = 4) and hyperglycaemic (DM, n = 8). Vimentin co-localised more frequently with glucagon (α-cells) than with insulin (β-cells) in the human ND group (1.43% total α-cells, 0.98% total β-cells, median; P < 0.05); these proportions were higher in T2DM than ND (median 4.53% α-, 2.53% β-cells; P < 0.05). Vimentin-positive β-cells were not apoptotic, had reduced expression of Nkx6.1 and Pdx1, and were not associated with islet amyloidosis or with bihormonal expression (insulin + glucagon). In non-human primates, vimentin-positive β-cell proportion was larger in the diabetic than the ND group (6.85 vs 0.50%, medians respectively, P < 0.05), but was similar in ND and HI groups. In conclusion, islet cell expression of vimentin indicates a degree of plasticity and dedifferentiation with potential loss of cellular identity in diabetes. This could contribute to α- and β-cell dysfunction in T2DM.

Foxa2 and Pdx1 cooperatively regulate postnatal maturation of pancreatic β-cells

ObjectiveThe transcription factors (TF) Foxa2 and Pdx1 are key regulators of beta-cell (β-cell) development and function. Mutations of these TFs or their respective cis-regulatory consensus binding sites have been linked to maturity diabetes of the young (MODY), pancreas agenesis, or diabetes susceptibility in human. Although Foxa2 has been shown to directly regulate Pdx1 expression during mouse embryonic development, the impact of this gene regulatory interaction on postnatal β-cell maturation remains obscure. MethodsIn order to easily monitor the expression domains of Foxa2 and Pdx1 and analyze their functional interconnection, we generated a novel double knock-in homozygous (FVFPBFDHom) fluorescent reporter mouse model by crossing the previously described Foxa2-Venus fusion (FVF) with the newly generated Pdx1-BFP (blue fluorescent protein) fusion (PBF) mice. ResultsAlthough adult PBF homozygous animals exhibited a reduction in expression levels of Pdx1, they are normoglycemic. On the contrary, despite normal pancreas and endocrine development, the FVFPBFDHom reporter male animals developed hyperglycemia at weaning age and displayed a reduction in Pdx1 levels in islets, which coincided with alterations in β-cell number and islet architecture. The failure to establish mature β-cells resulted in loss of β-cell identity and trans-differentiation towards other endocrine cell fates. Further analysis suggested that Foxa2 and Pdx1 genetically and functionally cooperate to regulate maturation of adult β-cells. ConclusionsOur data show that the maturation of pancreatic β-cells requires the cooperative function of Foxa2 and Pdx1. Understanding the postnatal gene regulatory network of β-cell maturation will help to decipher pathomechanisms of diabetes and identify triggers to regenerate dedifferentiated β-cell mass.