Foxo1 silencing triggers gut progenitors to differentiate into fully functional insulin-secreting cells that maintain glucose homeostasis.

Researchers have demonstrated that knocking out one gene in gut progenitor cells prompts them to differentiate into fully functional insulin-producing cells that can secrete insulin in response to changing blood glucose levels and maintain glucose homeostasis in diabetic mice. A team led by scientists at Columbia University’s Naomi Berrie Diabetes Center generated knockout mice lacking the Foxo1 transcription factor gene in Neurog3+ gut progenitor cells.

They found that these Foxo1-deficient progenitors differentiated into insulin-positive (Ins+) cells that expressed markers of mature β cells and secreted bioactive insulin as well as C-peptide in response to glucose. When the animals were treated with the pancreatic β cell toxin streptozotocin, the gut Ins+ cells regenerated and secreted insulin, reversing hyperglycemia in mice without the need for insulin therapy.

Domenico Accili, M.D., and colleagues claim their findings could lead to the development of new cell therapy approaches to treating diabetes. They report their work in Nature Genetics in a paper titled “Generation of functional insulin-producing cells in the gut by Foxo1 ablation.”

Research has demonstrated that a key step in the formation of endocrine cells is the generation of Neurog3-expressing (Neurog3+) cells from endodermal precursors, which then go on to differentiate into all known pancreatic islet cell types. Neurog3+ endocrine progenitors aren’t only located in the pancreas. Equivalent cells in the stomach and intestine generate most cells in the enteroendocrine system.

The Neurog3+ progenitors found in the pancreas and gut system share few properties, however, the authors point out. They give rise to cell types that have very different developmental fates and lifespans and which produce dissimilar peptide hormones. Moreover, pancreatic endocrine progenitors are formed almost exclusively during embryonic, whereas enteroendocrine progenitors continually arise from gut stem cells to enable population of endocrine gut cells.  

The Foxo family of transcription factors is known to play a key role in regulating both cell metabolism, and Foxo1 plays a role in the terminal differentiation of a range of cell types, the team continues. Foxo1 is co-expressed with Neurog3 in embryonic pancreas, and studies have shown that in human fetal pancreatic epithelium, knockdown of the Foxo1 protein increases the numbers of Neurog3+ cells.

To investigate the role of Foxo1 expression in the gut, the Columbia University researchers generated mice with somatic Foxo1 deletion in Neurog3+ enteroendocrine progenitors cells (Neurog3-Cre–driven Foxo1 knock­outs; NKO). They first confirmed that, as with the previous observations in Foxo1 protein-deficient human fetal pancreatic tissue, genetic knockdown of the gene in mice led to a marked increase in the numbers of Neurog3+ cells. They also found that Foxo1 knockdown inhibited Hes1, a gene that normally restricts the endocrine plasticity of Neurog3+ progenitors in endodermal development.

Interestingly, when the researchers analyzed the pancreas and intestines of newborn and adult NKO mice, they identified pancreatic hormone-producing cells in the neonatal gut, which included insulin-immunoreactive cells (Ins+) and cells immunoreactive with pancreatic glucagon (Gcg+) or polypeptide (Ppy+). Ins+ cells were also found throughout the gut of adult NKO animals, albeit in lower numbers than in neonates. In fact, knocking out Foxo1 in duodenal epithelial precursors, which are the cells that give rise to Neurog3+ cells, also resulted in increased Neurog3+ cells and the presence of Ins+ cells in the duodenum.

The gut Ins+ cells in NKO animals were found to display markers common to both insulin-producing and enteroendocryine cells as well as markers that are enriched specifically in pancreatic β cells but not in wild-type gut cells. The gut Ins+ cells were distinct from those producing Glp1, somatostatin, and Ppy, indicating that they weren’t mixed-lineage endocrine cells.

Importantly, ex vivo assays on matched gut tissue from both the NKO mice and wild-type mice indicated that the gut Ins+ cells were functional. NKO gut segments enriched for Ins+ cells released insulin and C-peptide in response to glucose, in a dose-dependent manner, possessed relevant sensing proteins, and were also sensitive to KATP channel blockers and openers. The intestinal insulin was also bioactive, as NKO gut extracts injected into newborn mice lowered blood glucose levels to the same degree as injections of recombinant human insulin. This could be blocked in both gut extract- and human insulin-treated animals by addition of an insulin-neutralizing antibody.

Because enteroendocrine cells are generated from Neurog3+ progenitors throughout life, unlike embryonic stem cell-derived pancreatic insulin-producing cells, the researchers hypothesized that gut Ins+ cells should demonstrate greater regenerative capacity than islet β cells in a streptozotocin (STZ)-induced mouse model of diabetes. Their studies showed that streptozotocin-induced diabetic wild-type mice needed daily insulin levels to survive due to pancreatic cell damage and all died on insulin withdrawal.

In contrast, glucose levels in the streptozotocin-induced diabetic NKO mice started to drop after insulin withdrawal and then stabilized, “consistent with the restoration of insulin production.” In fact 75% of NKO mice survived until the end of the experiments and also showed near-normal oral glucose tolerance, despite showing no evidence of pancreatic insulin production.

“All these findings make us think that coaxing a patient’s gut to make insulin-producing cells would be a better way to treat diabetes than therapies based on embryonic or induced pluripotent stem cells,” Dr. Accili remarks. The team in addition points out that such an approach should, feasibly, be safe. Their studies demonstrated that gut Ins+ cells release insulin in a glucose-regulated manner and that this insulin secretion can be inhibited by diazoxide, “allaying fears of unchecked insulin release that have plagued cellular replacement approaches to type 1 diabetes.”

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