Glucagon-producing α-cell transcriptional identity and reprogramming towards insulin production.

ß-Cell replacement by in situ reprogramming of non-ß-cells is a promising diabetes therapy. Following the observation that near-total ß-cell ablation in adult mice triggers the reprogramming of pancreatic α-, δ-, and γ-cells into insulin (INS)-producing cells, recent studies are delving deep into the mechanisms controlling adult α-cell identity. Systematic analyses of the α-cell transcriptome and epigenome have started to pinpoint features that could be crucial for maintaining α-cell identity. Using different transgenic and chemical approaches, significant advances have been made in reprogramming α-cells in vivo into INS-secreting cells in mice. The recent reprogramming of human α-cells in vitro is an important step forward that must now be complemented with a comprehensive molecular dissection of the mechanisms controlling α-cell identity.

Generation of human islet cell type-specific identity genesets.

Generation of surrogate cells with stable functional identities is crucial for developing cell-based therapies. Efforts to produce insulin-secreting replacement cells to treat diabetes require reliable tools to assess islet cellular identity. Here, we conduct a thorough single-cell transcriptomics meta-analysis to identify robustly expressed markers used to build genesets describing the identity of human α-, ß-, γ- and δ-cells. These genesets define islet cellular identities better than previously published genesets. We show their efficacy to outline cell identity changes and unravel some of their underlying genetic mechanisms, whether during embryonic pancreas development or in experimental setups aiming at developing glucose-responsive insulin-secreting cells, such as pluripotent stem-cell differentiation or in adult islet cell reprogramming protocols. These islet cell type-specific genesets represent valuable tools that accurately benchmark gain and loss in islet cell identity traits.

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.

Pancreatic Ppy-expressing γ-cells display mixed phenotypic traits and the adaptive plasticity to engage insulin production.

The cellular identity of pancreatic polypeptide (Ppy)-expressing γ-cells, one of the rarest pancreatic islet cell-type, remains elusive. Within islets, glucagon and somatostatin, released respectively from α- and δ-cells, modulate the secretion of insulin by ß-cells. Dysregulation of insulin production raises blood glucose levels, leading to diabetes onset. Here, we present the genetic signature of human and mouse γ-cells. Using different approaches, we identified a set of genes and pathways defining their functional identity. We found that the γ-cell population is heterogeneous, with subsets of cells producing another hormone in addition to Ppy. These bihormonal cells share identity markers typical of the other islet cell-types. In mice, Ppy gene inactivation or conditional γ-cell ablation did not alter glycemia nor body weight. Interestingly, upon ß-cell injury induction, γ-cells exhibited gene expression changes and some of them engaged insulin production, like α- and δ-cells. In conclusion, we provide a comprehensive characterization of γ-cells and highlight their plasticity and therapeutic potential.

A laboratory-scale binder jet additive manufacturing testbed for process exploration and material development.

Binder jet additive manufacturing (BJAM) is capable of fabricating complex three-dimensional components from a variety of material classes. Understanding the fundamentals of BJAM, including spreading of thin layers of powder, powder-binder interactions, and post-processing is critical to develop robust process parameters for BJAM. Toward meeting these needs, this work presents the design, fabrication, and qualification of a testbed for modular, mechanized, BJAM. The testbed seeks to replicate the operating conditions of commercial AM equipment and features fully programmable motion control including powder spreading using a precision roller mechanism, powder supply via a vibrating hopper, and gantry positioning of an inkjet printhead. The inkjet deposition system allows for the use of variable nozzle diameters, the exploration of novel binder compositions, and full control of jetting parameters. Validation of the accuracy and repeatability of the machine and its subsystems, as well as the fabrication of exemplary stainless steel components, are described. The precision engineered testbed can therefore enable the study of the BJAM process, exploration of novel binder compositions, and processing of custom powders to further scientific research and industrial applicability of BJAM.

Welding and Additive Manufacturing with Nanoparticle-Enhanced Aluminum 7075 Wire.

Aluminum alloy 7075 (Al 7075) with a T73 heat treatment is commonly used in aerospace applications due to exceptional specific strength properties. Challenges with manufacturing the material from the melt has previously limited the processing of Al 7075 via welding, casting, and additive manufacturing. Recent research has shown the capabilities of nanoparticle additives to control the solidification behavior of high-strength aluminum alloys, showcasing the first Al 7075 components processed via casting, welding, and AM. In this work, the properties of nanoparticle-enhanced aluminum 7075 are investigated on welded parts, overlays and through wire-based additive manufacturing. The hardness and tensile strength of the deposited materials were measured in the as-welded and T73 heat-treated conditions showing that the properties of Al 7075 T73 can be recovered in welded and layer-deposited parts. The work shows that Al 7075 now has the potential to be conventionally welded or additively manufactured from wire into high-strength, crack-free parts.

Combined inhibition of menin-MLL interaction and TGF-ß signaling induces replication of human pancreatic beta cells.

Both type 1 and type 2 diabetes are associated with hyperglycemia and loss of functional beta cell mass. Inducing proliferation of preexisting beta cells is an approach to increase the numbers of beta cells. In this study, we examined a panel of selected small molecules for their proliferation-inducing effects on human pancreatic beta cells. Our results demonstrated that a small molecule inhibitor of the menin-MLL interaction (MI-2) and small molecule inhibitors of TGF-ß signaling (SB431542, LY2157299, or LY364947) synergistically increased ex vivo replication of human beta cells. We showed that this increased proliferation did not affect insulin production, as a pivotal indication of beta cell function. We further provided evidence which suggested that menin-MLL and TGF-ß inhibition cooperated through downregulation of cell cycle inhibitors CDKN1A, CDKN1B, and CDKN2C. Our findings might provide a new option for extending the pharmacological repertoire for induction of beta cell proliferation as a potential therapeutic approach for diabetes.

Cell Heterogeneity and Paracrine Interactions in Human Islet Function: A Perspective Focused in ß-Cell Regeneration Strategies.

The ß-cell regeneration field has shown a strong knowledge boost in the last 10 years. Pluripotent stem cell differentiation and direct reprogramming from other adult cell types are becoming more tangible long-term diabetes therapies. Newly generated ß-like-cells consistently show hallmarks of native ß-cells and can restore normoglycemia in diabetic mice in virtually all recent studies. Nonetheless, these cells still show important compromises in insulin secretion, cell metabolism, electrical activity, and overall survival, perhaps due to a lack of signal integration from other islet cells. Mounting data suggest that diabetes is not only a ß-cell disease, as the other islet cell types also contribute to its physiopathology. Here, we present an update on the most recent studies of islet cell heterogeneity and paracrine interactions in the context of restoring an integrated islet function to improve ß-cell replacement therapies.

Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells.

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting ß-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-ß-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.

Pancreatic islet-autonomous insulin and smoothened-mediated signalling modulate identity changes of glucagon+ α-cells.

The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following ß-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of ß-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.

Estrogen Signals Through Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α to Reduce Oxidative Damage Associated With Diet-Induced Fatty Liver Disease.

BACKGROUND & AIMS: Inefficient fatty acid oxidation in mitochondria and increased oxidative damage are features of non-alcoholic fatty liver disease (NAFLD). In rodent models and patients with NAFLD, hepatic expression of peroxisome proliferator-activated receptor-γ (PPARG) coactivator 1α (PPARGC1A or PGC1A) is inversely correlated with liver fat and disease severity. A common polymorphism in this gene (rs8192678, encoding Gly482Ser) has been associated with NAFLD. We investigated whether reduced expression of PGC1A contributes to development of NAFLD using mouse models, primary hepatocytes, and human cell lines. METHODS: HepG2 cells were transfected with variants of PPARGC1A and protein and messenger RNA levels were measured. Mice with liver-specific hemizygous or homozygous disruption of Ppargc1a (Ppargc1af/+Alb-cre+/0 and Ppargc1af/f Alb-cre+/0 mice, respectively) were fed regular chow (control) or a high-fat diet supplemented with 30% d-fructose in drinking water (obesogenic diet) for 25-33 weeks. Liver tissues were analyzed by histology and by immunoblotting. Primary hepatocytes were analyzed for insulin signaling, reactive oxygen species, and estrogen response. Luciferase reporter expression was measured in transfected H2.35 cells expressing an estrogen receptor reporter gene, estrogen receptor 1, and/or PGC1A/B. RESULTS: The serine 482 variant of the human PGC1A protein had a shorter half-life than the glycine 482 variant when expressed in HepG2 cells. Liver tissues from mice with liver-specific hemizygous disruption of Ppargc1a placed on an obesogenic diet expressed increased markers of inflammation and fibrosis and decreased levels of antioxidant enzymes compared with the Ppargc1a+/+ on the same diet. Oxidative damage was observed in livers from Ppargc1af/+Alb-cre+/0 mice of each sex, in a cell-autonomous manner, but was greater in livers from the female mice. Expression of PGC1A in H2.35 cells coactivated estrogen receptor 1 and was required for estrogen-dependent expression of genes that encode antioxidant proteins. These findings could account for the increased liver damage observed in female Ppargc1af/+Alb-cre+/0 mice; while, compensatory increases in PPARG coactivator 1ß could prevent oxidative damage associated with complete loss of PGC1A expression in Ppargc1af/fAlb-cre+/0 female mice. CONCLUSIONS: In mice, loss of estrogen signaling contributes to oxidative damage caused by low levels of PGC1A in liver, exacerbating steatohepatitis associated with diets high in fructose and fat.

PGC-1 coactivators in ß-cells regulate lipid metabolism and are essential for insulin secretion coupled to fatty acids.

OBJECTIVES: Peroxisome proliferator-activated receptor γ coactivator 1 (PPARGCA1, PGC-1) transcriptional coactivators control gene programs important for nutrient metabolism. Islets of type 2 diabetic subjects have reduced PGC-1α expression and this is associated with decreased insulin secretion, yet little is known about why this occurs or what role it plays in the development of diabetes. Our goal was to delineate the role and importance of PGC-1 proteins to ß-cell function and energy homeostasis. METHODS: We investigated how nutrient signals regulate coactivator expression in islets and the metabolic consequences of reduced PGC-1α and PGC-1ß in primary and cultured ß-cells. Mice with inducible ß-cell specific double knockout of Pgc-1α/Pgc-1ß (ßPgc-1 KO) were created to determine the physiological impact of reduced Pgc1 expression on glucose homeostasis. RESULTS: Pgc-1α and Pgc-1ß expression was increased in primary mouse and human islets by acute glucose and palmitate exposure. Surprisingly, PGC-1 proteins were dispensable for the maintenance of mitochondrial mass, gene expression, and oxygen consumption in response to glucose in adult ß-cells. However, islets and mice with an inducible, ß-cell-specific PGC-1 knockout had decreased insulin secretion due in large part to loss of the potentiating effect of fatty acids. Consistent with an essential role for PGC-1 in lipid metabolism, ß-cells with reduced PGC-1s accumulated acyl-glycerols and PGC-1s controlled expression of key enzymes in lipolysis and the glycerolipid/free fatty acid cycle. CONCLUSIONS: These data highlight the importance of PGC-1s in coupling ß-cell lipid metabolism to promote efficient insulin secretion.

Phenotypic Characterization of MIP-CreERT1Lphi Mice With Transgene-Driven Islet Expression of Human Growth Hormone.

There is growing concern over confounding artifacts associated with ß-cell-specific Cre-recombinase transgenic models, raising questions about their general usefulness in research. The inducible ß-cell-specific transgenic (MIP-CreERT(1Lphi)) mouse was designed to circumvent many of these issues, and we investigated whether this tool effectively addressed concerns of ectopic expression and disruption of glucose metabolism. Recombinase activity was absent from the central nervous system using a reporter line and high-resolution microscopy. Despite increased pancreatic insulin content, MIP-CreERT mice on a chow diet exhibited normal ambient glycemia, glucose tolerance and insulin sensitivity, and appropriate insulin secretion in response to glucose in vivo and in vitro. However, MIP-CreERT mice on different genetic backgrounds were protected from high-fat/ streptozotocin (STZ)-induced hyperglycemia that was accompanied by increased insulin content and islet density. Ectopic human growth hormone (hGH) was highly expressed in MIP-CreERT islets independent of tamoxifen administration. Circulating insulin levels remained similar to wild-type controls, whereas STZ-associated increases in α-cell number and serum glucagon were significantly blunted in MIP-CreERT(1Lphi) mice, possibly due to paracrine effects of hGH-induced serotonin expression. These studies reveal important new insight into the strengths and limitations of the MIP-CreERT mouse line for ß-cell research.

Transient expression of Ngn3 in Xenopus endoderm promotes early and ectopic development of pancreatic beta and delta cells.

Promoting ectopic development of pancreatic beta cells from other cell types is one of the strategies being pursued for the treatment of diabetes. To achieve this, a detailed outline of the molecular lineage that operates in pancreatic progenitor cells to generate beta cells over other endocrine cell types is necessary. Here, we demonstrate that early transient expression of the endocrine progenitor bHLH protein Neurogenin 3 (Ngn3) favors the promotion of pancreatic beta and delta cell fates over an alpha cell fate, while later transient expression promotes ectopic development of all three endocrine cell fates. We found that short-term activation of Ngn3 in Xenopus laevis endoderm just after gastrulation was sufficient to promote both early and ectopic development of beta and delta cells. By examining gene expression changes 4 h after Ngn3 activation we identified several new downstream targets of Ngn3. We show that several of these are required for the promotion of ectopic beta cells by Ngn3 as well as for normal beta cell development. These results provide new detail regarding the Ngn3 transcriptional network operating in endocrine progenitor cells to specify a beta cell phenotype and should help define new approaches to promote ectopic development of beta cells for diabetes therapy.