Successful in vitro fertilization and generation of transgenics in Black and Tan Brachyury (BTBR) mice.

The Black and Tan Brachyury (BTBR) mouse strain is a valuable model for the study of long-term complications from obesity-induced type 2 diabetes mellitus and autism spectrum disorder. Due to technical difficulties with assisted reproduction, genetically modified animals on this background have previously been generated through extensive backcrossing, which is expensive and time-consuming. We successfully generated two separate transgenic mouse lines after direct zygote microinjection into this background strain. Additionally, we developed in vitro fertilization (IVF) methods for the BTBR mouse. We found low rates of fertilization and implantation in this strain, and identified the BTBR oocyte as the primary culprit of low success with BTBR IVF. We achieved an increase in live born pups from 5.9 to 35.6 % with IVF in the BTBR strain by use of BTBR females at a younger age (18-25 days), collection of oocytes 15-17 h after superovulation, and the use of supplemented fertilization media. This method eliminates the need for time consuming assisted embryo manipulations that are otherwise required for success with BTBR oocytes. This advancement provides an exciting opportunity to directly generate BTBR transgenics and gene-edited mice using both traditional and emerging genomic editing techniques, such as CRISPR/Cas9. These methods also allow effective colony preservation and rederivation with these strains. To our knowledge, this is the first report describing embryo manipulations in BTBR mice.

Tcf19 is a novel islet factor necessary for proliferation and survival in the INS-1 ß-cell line.

Recently, a novel type 1 diabetes association locus was identified at human chromosome 6p31.3, and transcription factor 19 (TCF19) is a likely causal gene. Little is known about Tcf19, and we now show that it plays a role in both proliferation and apoptosis in insulinoma cells. Tcf19 is expressed in mouse and human islets, with increasing mRNA expression in nondiabetic obesity. The expression of Tcf19 is correlated with ß-cell mass expansion, suggesting that it may be a transcriptional regulator of ß-cell mass. Increasing proliferation and decreasing apoptotic cell death are two strategies to increase pancreatic ß-cell mass and prevent or delay diabetes. siRNA-mediated knockdown of Tcf19 in the INS-1 insulinoma cell line, a ß-cell model, results in a decrease in proliferation and an increase in apoptosis. There was a significant reduction in the expression of numerous cell cycle genes from the late G1 phase through the M phase, and cells were arrested at the G1/S checkpoint. We also observed increased apoptosis and susceptibility to endoplasmic reticulum (ER) stress after Tcf19 knockdown. There was a reduction in expression of genes important for the maintenance of ER homeostasis (Bip, p58(IPK), Edem1, and calreticulin) and an increase in proapoptotic genes (Bim, Bid, Nix, Gadd34, and Pdia2). Therefore, Tcf19 is necessary for both proliferation and survival and is a novel regulator of these pathways.

Cholecystokinin attenuates ß-cell apoptosis in both mouse and human islets.

Loss of functional pancreatic ß-cell mass and increased ß-cell apoptosis are fundamental to the pathophysiology of type 1 and type 2 diabetes. Pancreatic islet transplantation has the potential to cure type 1 diabetes but is often ineffective due to the death of the islet graft within the first few years after transplant. Therapeutic strategies to directly target pancreatic ß-cell survival are needed to prevent and treat diabetes and to improve islet transplant outcomes. Reducing ß-cell apoptosis is also a therapeutic strategy for type 2 diabetes. Cholecystokinin (CCK) is a peptide hormone typically produced in the gut after food intake, with positive effects on obesity and glucose metabolism in mouse models and human subjects. We have previously shown that pancreatic islets also produce CCK. The production of CCK within the islet promotes ß-cell survival in rodent models of diabetes and aging. We demonstrate a direct effect of CCK to reduce cytokine-mediated apoptosis in a ß-cell line and in isolated mouse islets in a receptor-dependent manner. However, whether CCK can protect human ß-cells was previously unknown. Here, we report that CCK can also reduce cytokine-mediated apoptosis in isolated human islets and CCK treatment in vivo decreases ß-cell apoptosis in human islets transplanted into the kidney capsule of diabetic NOD/SCID mice. Collectively, these data identify CCK as a novel therapy that can directly promote ß-cell survival in human islets and has therapeutic potential to preserve ß-cell mass in diabetes and as an adjunct therapy after transplant.

TCF19 Impacts a Network of Inflammatory and DNA Damage Response Genes in the Pancreatic ß-Cell.

Transcription factor 19 (TCF19) is a gene associated with type 1 diabetes (T1DM) and type 2 diabetes (T2DM) in genome-wide association studies. Prior studies have demonstrated that Tcf19 knockdown impairs ß-cell proliferation and increases apoptosis. However, little is known about its role in diabetes pathogenesis or the effects of TCF19 gain-of-function. The aim of this study was to examine the impact of TCF19 overexpression in INS-1 ß-cells and human islets on proliferation and gene expression. With TCF19 overexpression, there was an increase in nucleotide incorporation without any change in cell cycle gene expression, alluding to an alternate process of nucleotide incorporation. Analysis of RNA-seq of TCF19 overexpressing cells revealed increased expression of several DNA damage response (DDR) genes, as well as a tightly linked set of genes involved in viral responses, immune system processes, and inflammation. This connectivity between DNA damage and inflammatory gene expression has not been well studied in the ß-cell and suggests a novel role for TCF19 in regulating these pathways. Future studies determining how TCF19 may modulate these pathways can provide potential targets for improving ß-cell survival.

Enriching Islet Phospholipids With Eicosapentaenoic Acid Reduces Prostaglandin E2 Signaling and Enhances Diabetic ß-Cell Function.

Prostaglandin E2 (PGE2) is derived from arachidonic acid, whereas PGE3 is derived from eicosapentaenoic acid (EPA) using the same downstream metabolic enzymes. Little is known about the impact of EPA and PGE3 on ß-cell function, particularly in the diabetic state. In this work, we determined that PGE3 elicits a 10-fold weaker reduction in glucose-stimulated insulin secretion through the EP3 receptor as compared with PGE2 We tested the hypothesis that enriching pancreatic islet cell membranes with EPA, thereby reducing arachidonic acid abundance, would positively impact ß-cell function in the diabetic state. EPA-enriched islets isolated from diabetic BTBR Leptinob/ob mice produced significantly less PGE2 and more PGE3 than controls, correlating with improved glucose-stimulated insulin secretion. NAD(P)H fluorescence lifetime imaging showed that EPA acts downstream and independently of mitochondrial function. EPA treatment also reduced islet interleukin-1ß expression, a proinflammatory cytokine known to stimulate prostaglandin production and EP3 expression. Finally, EPA feeding improved glucose tolerance and ß-cell function in a mouse model of diabetes that incorporates a strong immune phenotype: the NOD mouse. In sum, increasing pancreatic islet EPA abundance improves diabetic ß-cell function through both direct and indirect mechanisms that converge on reduced EP3 signaling.

Attention to Background Strain Is Essential for Metabolic Research: C57BL/6 and the International Knockout Mouse Consortium.

The International Knockout Mouse Consortium (IKMC) introduces its targeted constructs into C57BL/6N embryonic stem cells. However, breeding with a Cre-recombinase and/or Flp-recombinase mouse is required for the generation of a null allele with the IKMC cassette. Many recombinase strains are in the C57BL/6J background, resulting in knockout animals on a mixed strain background. This can lead to variability in metabolic data and the use of improper control groups. While C57BL/6N and C57BL/6J are derived from the same parental C57BL/6 strain, there are key genotypic and phenotypic differences between these substrains. Many researchers may not even be aware of these differences, as the shorthand C57BL/6 is often used to describe both substrains. We found that 58% of articles involving genetically modified mouse models did not completely address background strain. This review will describe these two substrains and highlight the importance of separate consideration in mouse model development. Our aim is to increase awareness of this issue in the diabetes research community and to provide practical strategies to enable researchers to avoid mixed strain animals when using IKMC knockout mice.