Regulation of pancreatic beta cell proliferation and aging

The replicative capacity of insulin-producing pancreatic beta cells is dynamically regulated during development, maturation, and aging. Early in life, beta cells proliferate rapidly to expand beta cell mass but quickly become quiescent with age. While this decline is well documented, the mechanisms that underlie this age-dependent beta cell replicative senescence are still poorly understood. Using mouse genetics and in vivo quantitative proteomics approaches, we found that nutrient sensing plays an important role in controlling the proliferation of beta cells. We show that the transcription factor Nkx6.1 is required for expanding beta cell mass during the early wave of rapid postnatal beta cell proliferation by regulating the expression of the nutrient sensing receptors Glut2 and Glp1r. Furthermore, by quantitatively comparing the proteome of islets from young and aged mice, we found dynamic regulation of not only cell cycle proteins, but also proteins critical for beta cell function. Proteins important for insulin secretion and metabolic regulation increased with age, while proteins involved in expanding cell number declined with age. From our proteomic screen, we identified a protein deacetylase upregulated during aging. Pharmacologic inhibition of this protein promoted both rodent and human beta cell proliferation ex vivo, indicating the deacetylation activity of this candidate represses beta cell proliferation. Beta cell-specific deletion of the protein deacetylase increased rodent beta cell proliferation and mass in diabetic mice in vivo. Importantly, inhibition of this protein in human islets ex vivo did not negatively affect beta cell function or survival. Finally, we show that the protein deacetylase specifically regulates beta cell proliferation in conditions of elevated blood glucose through modulating the glucose-dependent MAPK pathway. Overall, our studies have uncovered dynamic regulation of beta cell proliferation from birth to advanced age and identified a viable therapeutic target for enhancing beta cell mass for the treatment of diabetes

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

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