Advances in β cell replacement and regeneration strategies for treating diabetes

In the past decade, new approaches have been explored that are aimed at restoring functional β cell mass as a treatment strategy for diabetes. The two most intensely pursued strategies are β cell replacement through conversion of other cell types and β cell regeneration by enhancement of β cell replication. The approach closest to clinical implementation is the replacement of β cells with human pluripotent stem cell-derived (hPSC-derived) cells, which are currently under investigation in a clinical trial to assess their safety in humans. In addition, there has been success in reprogramming developmentally related cell types into β cells. Reprogramming approaches could find therapeutic applications by inducing β cell conversion in vivo or by reprogramming cells ex vivo followed by implantation. Finally, recent studies have revealed novel pharmacologic targets for stimulating β cell replication. Manipulating these targets or the pathways they regulate could be a strategy for promoting the expansion of residual β cells in diabetic patients. Here, we provide an overview of progress made toward β cell replacement and regeneration and discuss promises and challenges for clinical implementation of these strategies

Advances in β cell replacement and regeneration strategies for treating diabetes

In the past decade, new approaches have been explored that are aimed at restoring functional β cell mass as a treatment strategy for diabetes. The two most intensely pursued strategies are β cell replacement through conversion of other cell types and β cell regeneration by enhancement of β cell replication. The approach closest to clinical implementation is the replacement of β cells with human pluripotent stem cell–derived (hPSC-derived) cells, which are currently under investigation in a clinical trial to assess their safety in humans. In addition, there has been success in reprogramming developmentally related cell types into β cells. Reprogramming approaches could find therapeutic applications by inducing β cell conversion in vivo or by reprogramming cells ex vivo followed by implantation. Finally, recent studies have revealed novel pharmacologic targets for stimulating β cell replication. Manipulating these targets or the pathways they regulate could be a strategy for promoting the expansion of residual β cells in diabetic patients. Here, we provide an overview of progress made toward β cell replacement and regeneration and discuss promises and challenges for clinical implementation of these strategies

Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin(+) cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)-derived insulin(+) cells, our study has significant implications for developing cell replacement therapies

Another puzzle piece: new record of the Fringed Leaf Frog, Cruziohyla craspedopus (Funkhouser, 1957) (Anura: Phyllomedusidae), in the eastern Amazon Rainforest

Pancreatic β cell physiology changes substantially throughout life, yet the mechanisms that drive these changes are poorly understood. Here, we performed comprehensive in vivo quantitative proteomic profiling of pancreatic islets from juvenile and 1-year-old mice. The analysis revealed striking differences in abundance of enzymes controlling glucose metabolism. We show that these changes in protein abundance are associated with higher activities of glucose metabolic enzymes involved in coupling factor generation as well as increased activity of the coupling factor-dependent amplifying pathway of insulin secretion. Nutrient tracing and targeted metabolomics demonstrated accelerated accumulation of glucose-derived metabolites and coupling factors in islets from 1-year-old mice, indicating that age-related changes in glucose metabolism contribute to improved glucose-stimulated insulin secretion with age. Together, our study provides an in-depth characterization of age-related changes in the islet proteome and establishes metabolic rewiring as an important mechanism for age-associated changes in β cell function

Integrated In Vivo Quantitative Proteomics and Nutrient Tracing Reveals Age-Related Metabolic Rewiring of Pancreatic β Cell Function.

Pancreatic β cell physiology changes substantially throughout life, yet the mechanisms that drive these changes are poorly understood. Here, we performed comprehensive in vivo quantitative proteomic profiling of pancreatic islets from juvenile and 1-year-old mice. The analysis revealed striking differences in abundance of enzymes controlling glucose metabolism. We show that these changes in protein abundance are associated with higher activities of glucose metabolic enzymes involved in coupling factor generation as well as increased activity of the coupling factor-dependent amplifying pathway of insulin secretion. Nutrient tracing and targeted metabolomics demonstrated accelerated accumulation of glucose-derived metabolites and coupling factors in islets from 1-year-old mice, indicating that age-related changes in glucose metabolism contribute to improved glucose-stimulated insulin secretion with age. Together, our study provides an in-depth characterization of age-related changes in the islet proteome and establishes metabolic rewiring as an important mechanism for age-associated changes in β cell function

<i>Nkx6.1</i> is required for beta cell specification downstream of Ngn3.

<p>(A, B) Schematic of the alleles and transgenes for <i>Nkx6.1</i> inactivation and lineage tracing; Triangles, <i>loxP</i> sites. Immunofluorescence staining of pancreata at e15.5 (C, D) or postnatal day (P) 2 (E–P). Recombined, GFP⁺ cells are restricted to the endocrine compartment (antibody against the pan-endocrine marker Chromogranin A, Chga) in control (E) and <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> mice (F). The insets show higher magnifications and arrowheads point to GFP⁺ cells expressing Ngn3 (C, D) or hormones (G–P). Quantification of hormone⁺GFP⁺ (Q), Ki67⁺GFP⁺ (R), or TUNEL⁺GFP⁺ (S) co-positive cells as a percentage of all GFP-expressing cells in pancreata of <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> and <i>Ngn3-Cre;Z/EG</i> mice at P2 (n = 4). Loss of <i>Nkx6.1</i> in endocrine precursors favors alternative, non-beta endocrine cell fate choices over beta cell fate. Horm, hormones; Ins, insulin; Glc, glucagon; Som, somatostatin; PP, pancreatic polypeptide; Ghr, ghrelin; endo, endocrine. Scale bar = 50 µm. Error bars represent S.E.M; *p<0.05, **p<0.01.</p

Nkx6.1 and Isl1 function as antagonistic transcriptional regulators of the <i>Arx Re1</i> enhancer.

<p>Immunofluorescence staining of pancreata from <i>Ngn3-Cre;Z/EG</i> mice at e14.5 (A) and e16.5 (B) for Nkx6.1, Arx, and GFP shows that the majority of progeny of Ngn3-expressing cells (GFP⁺) co-express Arx and Nkx6.1 at e14.5 (arrowheads in A), while the Arx⁺ and Nkx6.1⁺ domains are distinct at e16.5 (arrowheads in B point to GFP⁺Arx⁺Nkx6.1⁻ cells). (C) Schematic of the <i>Arx</i> locus; asterisks indicate phylogenetically-conserved Nkx6.1 binding motifs and numbers indicate the distance from the transcriptional start site. Nkx6.1 binds to site C (<i>Re1</i> element) in the <i>Arx</i> locus in chromatin from Min6 cells (D) and FACS-sorted GFP⁺ cells (E) from e15.5 pancreata of <i>Neurog3</i>^eGFP embryos analyzed by ChIP with antibodies against Nkx6.1 or control immunoglobulin G (IgG). Mouse <i>glucagon</i> promoter and intergenic primers were used as positive (+) and negative (−) controls, respectively. (F) Co-transfection of αTC1–6 cells with the <i>Arx Re1</i> enhancer-luciferase construct, the <i>CMV-Renilla</i> expression construct, and with or without the <i>CMV-Nkx6.1</i> expression construct. Lane one (M) represents basal luciferase expression of the minimal promoter. Luciferase activity was quantified relative to the expression of the minimal promoter. Activity of the <i>Re1</i> enhancer is repressed by Nkx6.1. (G) Co-transfection of αTC1–6 cells with the <i>Arx Re1</i> enhancer-luciferase construct, <i>CMV-Renilla</i>, and with different concentration of <i>CMV-Nkx6.1</i> and <i>CMV-Isl1</i>, as indicated. Nkx6.1 prevents activation of the <i>Arx Re1</i> enhancer by Isl1 in a dose-dependent manner (lanes 2–7). Luciferase activity was quantified relative to the expression of the <i>Re1</i> enhancer. Increasing concentrations of Isl1 are not sufficient to restore baseline activity of the <i>Re1</i> enhancer in the presence of 200 ng of <i>CMV-Nkx6.1</i> (lanes 8–12). Scale bar = 50 µm. Error bars represent S.E.M; *p<0.05, **p<0.01, ***p<0.001.</p

Loss of <i>Nkx6.1</i> in endocrine precursors results in activation of non-beta endocrine genes.

<p>(A–L) Immunofluorescence staining of pancreata from <i>Ngn3-Cre;Z/EG</i> and <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> mice at postnatal day (P) 2 shows reduced Pdx1 (A, B), absence of MafA (C, D), and ectopic expression of Arx (E, F), Brn4 (G, H), glucagon (Glc; I, J), and somatostatin (Som; K, L) in <i>Nkx6.1</i>-deficient, recombined, insulin⁺GFP⁺ cells. Arrowheads point to insulin⁺ cells ectopically expressing non-beta endocrine markers. Ins, insulin. Scale bar = 50 µm.</p

Loss of <i>Nkx6.1</i> in beta cells causes beta-to-delta cell conversion.

<p>Immunofluorescence staining of pancreata from <i>Nkx6.1^f/+;RIP-Cre;R26-YFP</i> and <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> mice at 6 weeks of age shows Nkx6.1 (A) and insulin (B) expression in YFP⁺ cells of <i>Nkx6.1^f/+;RIP-Cre;R26-YFP</i> control mice, but loss of Nkx6.1 (F) and insulin (G) in YFP⁺ cells of <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> mice. The insets display higher magnification images. YFP⁺ cells do not express glucagon (C, H) and rarely express pancreatic polypeptide (E, J; insets, arrowheads) in either genotype. While YFP⁺ cells are somatostatin⁻ in <i>Nkx6.1^f/+;RIP-Cre;R26-YFP</i> mice (D; insets, arrowheads), YFP-labeled cells are mostly somatostatin⁺ in <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> mice (I; insets, arrowheads), suggesting beta-to-delta cell conversion. Arx expression is similar in both genotypes and absent from lineage-labeled YFP⁺ cells (K, M; inset, arrowhead), showing that loss of <i>Nkx6.1</i> in beta cells does not activate <i>Arx</i>. Pdx1⁺somatostatin⁺ cells are found in both genotypes (L, N; insets, arrowheads), but express YFP only in <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> mice (L; inset, arrowhead). (O) Quantification of the percentage of hormone⁺YFP⁺ cells relative to all hormone⁺ cells for each islet cell type shows reduced numbers of insulin⁺YFP⁺ cells and increased numbers of in somatostatin⁺YFP⁺ cells in <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> mice compared to <i>Nkx6.1^f/+;RIP-Cre;R26-YFP</i> mice at 6 weeks (n = 3). Wks, weeks; Ins, insulin; Glc, glucagon; PP, pancreatic polypeptide; Som, somatostatin; Horm, hormones. Scale bar = 50 µm. Error bars represent S.E.M; ***p<0.0001.</p